Modulators of proteins with phosphotryrosine recognition units

ABSTRACT

Y--X--C(R&#39;)═C(R&#34;)COOR&#39;&#34;                                (A1) 
    
     The present invention relates to novel protein tyrosine phosphatase modulating compounds having the general structure shown in Formula (A1), to methods for their preparation, to compositions comprising the compounds, to their use for treatment of human and animal disorders, to their use for purification of proteins or glycoproteins, and to their use in diagnosis. The invention relates to modulation of the activity of molecules with phosphotyrosine recognition units, including protein tyrosine phosphatases (PTPases) and proteins with Src-homology-2 domains, in in vitro systems, microorganisms, eukaryoic cells, whole animals and human beings. R&#39; and R&#34; are independently selected from the group consisting of hydrogen, halo, cyano, nitro, trihalomethyl, alkyl, arylalkyl. R&#39;&#34; is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, arylalkyl. X is aryl. Y is selected from hydrogen or ##STR1## wherein (*) indicates a potential point of attachment to X.

This application is a continuation-in-part-application of applicationSer. No. 08/543,630 filed Oct. 16th, 1995, now pending, which in turnclaims the benefit of the filing date of application Ser. No. 60/017,610filed Jun. 19th, 1995, now abandoned.

FIELD OF THE INVENTION

The present invention relates to novel protein tyrosine phosphatasemodulating compounds, to methods for their preparation, to compositionscomprising the compounds, to their use for treatment of human and animaldisorders, to their use for purification of proteins or glycoproteins,and to their use in diagnosis. The invention relates to modulation ofthe activity of molecules with phosphotyrosine recognition units,including protein tyrosine phosphatases (PTPases) and proteins withSrc-homology-2 domains, in in vitro systems, microorganisms, eukaryoiccells, whole animals and human beings.

BACKGROUND OF THE INVENTION

Reversible phosphorylation of proteins is a prevalent biologicalmechanism for modulation of enzymatic activity in living organisms.Tonks et al., J. Biol. Chem., 263(14):6722-30 (1988). Such reversiblephosphorylation requires both a protein kinase (PK), to phosphorylate aprotein at a particular amino acid residue, and a protein phosphatase(PP), to remove the phosphate moieties. See generally, Hunter, Cell,80:225-236 (1995). Recently, it has been estimated that humans have asmany as 2000 conventional PK genes, and as many as 1000 PP genes. Id.

One major class of PK's/PP's--the protein serine/threonine kinases andprotein serine/threonine phosphatases--have been shown to play criticalroles in the regulation of metabolism. See generally, Cohen, TrendsBiochem. Sci., 17:408-413 (1992); Shenolikar, Ann. Rev. Cell Biol.,10:55-86 (1994); Bollen et al., Crit. Rev. Biochem. Mol. Biol.,27:227-81 (1992). As their name suggests, these enzymes phosphorylateand dephoshphorylate serine or threonine residues of substrate proteins.Inhibitors of protein serine/threonine phosphatases and kinases havebeen described. See, e.g., MacKintosh and MacKintosh, TIBS, 19:444-448(1994).

The protein tyrosine kinases/phosphatases comprise a second, distinctfamily of PK/PP enzymes of significant interest, and have beenimplicated in the control of normal and neoplastic cell growth andproliferation. See Fisher et al., Science, 253:401-406 (1991). Proteintyrosine kinase (PTK) genes are ancient in evolutionary origin and sharea high degree of inter-species conservation. See generally Hunter andCooper, Ann. Rev. Biochem., 54:897-930 (1985). PTK enzymes exhibit highspecificity for tyrosine, and ordinarily do not phosphorylate serine,threonine, or hydroxyproline.

More than 75 members of the PTPase family have been identified ineukaryotes, prokaryotes, and even viruses. Tonks and Neel, Cell87:365-368. Protein tyrosine phosphatases (PTPases) were originallyidentified and purified from cell and tissue lysates using a variety ofartificial substrates, and therefore their natural functions andsubstrates were not obvious. However, their roles in cellular processes,including cell-cell contact and cell adhesion, and growth factor andantigen signaling events, have begun to be elucidated.

PTPases are generally grouped into two categories: those which have bothan extracellular domain and an intracellular catalytic domain, thereceptor PTPases (R-PTPases); and those which are entirelyintracellular. For R-PTPases much effort has been directed atdetermining the function of the extracellular domain. Most of theR-PTPases contain extracellular domains which are structurally similarto domains found in known adhesion molecules; these domains includefibronectin type III repeats, immunoglobulin domains, and cadherinextracellular repeats. See generally Brady-Kalnay and Tonks, Curr. Opin.Cell. Biol. 7:650-657 (1995); Streuli, Curr. Opin. Cell. Biol. 8:182-188(1996). This homology with proteins known to be involved in adhesionsuggested a role for these R-PTPases in regulating or mediating adhesionevents. For several of the R-PTPases, this has now been demonstrated.

Cells form specialized structures at the sites of cell-cell contact(adherens junctions) and cell-extracellular matrix contact (focaladhesion). Multiple signal transduction molecules are recruited to thesesites, including several PTK's; and these sites are characterized byincreased protein tyrosine phosphorylation. These sites are impermanent,and are created and destroyed as required for cell mobility. As enhancedtyrosine phosphorylation is characteristic of the formation of adherensjunctions and focal adhesions, it is likely that protein tyrosinedephosphorylation by PTPases serves to regulate the creation anddestruction of the sites. Supporting this, several studies have shownthat treatment with a general PTPase inhibitor (vanadate) resulted inincreased focal adhesion formation and increased cell spreading. Volberget al., The EMBO J. 11:1733-1742 (1992); Bennett et al., J. Cell Sci.106:891-901 (1993). Importantly, the broadly-expressed LAR R-PTPase hasbeen demonstrated to localize to focal adhesions, apparently via theLAR-interacting protein LIP.1. Serra-Pages et al., The EMBO J.14:2827-2838 (1995). As PTPδ and PTPσ, both R-PTPases, also associatewith LIP.1 Pulido et al., Proc. Natl. Acad. Sci. 92:11686-11690 (1995)!,it is likely that these two phosphatases can also localize to focaladhesions. Most significantly, LAR only localized to the portion of thefocal adhesion which is proximal to the nucleus, and is thought to beundergoing disassembly. Thus it is likely that these phosphatases act tonegatively regulate focal adhesion formation, acting to enhance thedestruction of the focal adhesion site.

R-PTPases may also act to positively regulate adhesion. Adherensjunctions contain, among others, adhesion receptors termed cadherinswhich mediate cell-cell contact through homophilic binding; thecadherins associate with α-, β-, and γ-catenins, intracellular proteinswhich interact with cortical actin. Association between cadherins andcatenins serves to stabilize the adherens junction and to strengthencell-cell contact. See generally Cowin, Proc. Natl. Acad. Sci.91:10759-10761 (1994). Association of cadherin with β-catenin isdecreased by tyrosine phosphorylation of β-catenin Kinch et al., J.Cell. Biol. 130:461-471 (1995); Behrens et al., J. Cell. Biol.120:757-766 (1993)!; moreover, treatment with the PTPase inhibitorvanadate inhibits cadherin-dependent adhesion Matsuyoshi et al., J.Cell. Biol. 118:703-714 (1992)!. Collectively, these data indicate thatPTPase activity is critical in maintaining cadherin-mediated cellaggregation. The R-PTPases PTPμ and PTPκ associate intracellularly withcadherins, and colocalize with cadherins and catenins to adherensjunctions Brady-Kalnay et al., J. Cell. Biol. 130:977-986 (1995); Fuchset al., J. Biol. Chem. 271:16712-16719 (1996)!; thus PTPμ and PTPκ arelikely to enhance cadherin function by limiting catenin phosphorylation.

In addition to their catalytic function in regulating adhesion events,several R-PTPases have direct roles in mediating adhesion through theirextracellular domains. PTPκ and PTPμ mediate cellular aggregationthrough homophilic binding Brady-Kalnay et al., J. Cell. Biol.122:961-972 (1993); Gebbink et al., J. Biol. Chem. 268:16101-16104(1993); Sap et al., Mol. Cell. Biol. 14:1-9 (1994)!. The neuronal PTPζ(which has also been called R-PTPβ) binds to contactin, a neuronal cellrecognition molecule; binding of PTPζ to contactin increases celladhesion and neurite outgrowth. Peles et al., Cell 82:251-260 (1995). Asecreted splice variant of PTPζ (also known as phosphacan) binds theextracellular matrix protein tenascin Barnea et al. J. Biol. Chem.269:14349-14352 (1994)!, and the neural cell adhesion molecules N-CAMand Ng-CAM Maurel et al., Proc. Natl. Acad. Sci. 91:2512-2516 (1994)!.As the expression of PTPζ is restricted to radial glial cells in thedeveloping central nervous system, which are though to form barriers toneuronal migration during embryogenesis, it is likely that theinteraction of PTPζ with contactin, tenascin, N-CAM, and/or Ng-CAM actsto regulate neuronal migration. This has been demonstrated for a relatedR-PTPase, DLAR, in Drosophila Krueger et al. Cell 84:611-622 (1996)!.

Because tyrosine phosphorylation by PTK enzymes usually is associatedwith cell proliferation, cell transformation and cell differentiation,it was assumed that PTPases were also associated with these events. Forseveral of the intracellular PTPases, this function has now beenverified.

SHP1 (which has also been called SHPTP1, SHP, HCP, and PTP-1C see Adachiet al., Cell 85:15 (1996)!), an intracellular PTPase which contains twoamino-terminal phosphotyrosyl binding Src Homology 2 (SH2) domainsfollowed by the catalytic PTPase domain, has been demonstrated to be animportant negative regulator of growth factor signaling events. Seegenerally Tonks and Neel, supra; Streuli, supra. In mice, loss of SHP1function (the motheaten and viable motheaten phenotypes) causes multiplehematopoietic defects resulting in immunodeficiency and severeautoimmunity; culminating in lethality by 2-3 weeks or 2-3 monthsdepending on the severity of SHP1 deficiency. Although these mice havereduced numbers of hematopoietic cells, suggesting defects indevelopment and maturation, those cells which survive and enter theperiphery are characterized by hyper-responsiveness to growth factorsand antigen. This observation suggested a role for SHP1 in negativeregulation of hematopoietic signaling events.

This has now been well-established for the erythropoietin receptor(EpoR), a member of the cytokine receptor family (which also includesthe receptors for interleukins 2, 3, 4, 5, 6, 7; granulocyte-macrophagecolony stimulating factor, and macrophage colony stimulating factor).SHP1 associates via its SH2 domains with tyrosine-phosphorylated EpoR,causing dephosphorylation and inactivation of the EpoR-associated Januskinase 2 and termination of the cellular response to erythropoietin.Klingmuller et al., Cell 80:729-738 (1995). Mutation of the tyrosine onthe EpoR to which SHP1 binds results in enhanced cell proliferation toerythropoietin in vitro Klingmuller, supra!. In humans, mutation of theEpoR resulting in loss of association with SHP1 causes autosomaldominant benign erythrocytosis, which is characterized by increasednumbers of erythrocytes in the periphery and increased hematocrit. de laChapelle et al., Proc. Natl. Acad. Sci. 90:4495-4499 (1993).

SHP1 also appears to be a negative regulator of the cellular response tocolony stimulating factor-1 (CSF-1, a major macrophage mitogeniccytokine), as cells from viable motheaten and motheaten mice, which havereduced or absent SHP1 function, are hyper-responsive to CSF-1 in vitro.Reduced SHP1 expression also results in increased cellular response tointerleukin 3 Yi et al., Mol. Cell. Biol. 13:7577-7586 (1993)!.Collectively, these observations suggest that SHP1 functions to limitthe cellular response to cytokines and growth factors by reversing thetyrosine phosphorylation of key signaling intermediates in thesepathways.

PTPases appear to play a homologous role in the insulin signalingpathway. Treatment of adipocytes with the PTPase inhibitor vanadateresults in increased tyrosine phosphorylation and tyrosine kinaseactivity of the insulin receptor (InsR), and enhances or mimics thecellular effects of insulin including increased glucose transport. See,e.g., Shisheva and Shechter, Endocrinology 133:1562-1568 (1993); Fantus,et al., Biochemistry 28:8864-8871 (1989); Kadota, et al., Biochem.Biophys. Res. Comm. 147:259-266 (1987); Kadota, et al., J. Biol. Chem.262:8252-8256 (1987). Transiently induced reduction in expression of twoPTPases, the intracellular PTPase PTP-1B and the R-PTPase LAR, resultedin similar increases in the cellular response to insulin. Kulas, et al.,J. Biol. Chem. 270:2435-2438 (1995); Ahmad et al., J. Biol. Chem.270:20503-20508 (1995). Conversely, increased cellular expression ofseveral PTPases (PTPα, PTPε, CD45) in vitro has been demonstrated toresult in diminished InsR signaling see, e.g., Moller, et al., J. Biol.Chem. 271:23126-23131 (1995); Kulas et al., J. Biol. Chem. 271:755-760(1996)!. Finally, increased expression of LAR was observed in adiposetissue from obese human subjects Ahmad, et al., J. Clin. Invest.95:2806-2812 (1995)!. These data provide clear evidence that PTPasesnegatively regulate the insulin signaling pathway.

While many of the PTPases function to negatively regulate cellularmetabolism and response, it is becoming increasingly evident thatPTPases provide important positive signaling mechanisms as well. Perhapsthe best example of such a positive regulator is the hematopoieticR-PTPase CD45. See generally Streuli, supra; Okumura and Thomas, supra;Trowbridge, Annu. Rev. Immunol. 12:85-116 (1994). CD45 is abundantlyexpressed on the cell surface of all nucleated hematopoietic cells, inseveral alternative splice variants. T and B lymphocytes which lack CD45expression are incapable of responding normally to antigen, suggestingthat CD45 is required for antigen receptor signaling. Geneticallyengineered mice which lack expression of CD45 exhibit severe defects inT lymphocyte development and maturation, indicating an additional rolefor CD45 in thymopoiesis. The major substrates for CD45 appear to bemembers of the Src family of PTK's, particularly Lck and Fyn, whosekinase activity is both positively and negatively regulated by tyrosinephosphorylation. Lck and Fyn isolated from CD45-deficient cells arehyperphosphorylated on negative regulatory tyrosine residues, and theirPTK activity is reduced. As CD45 can dephosphorylate and activatepurified Lck and Fyn in vitro, these data suggest that CD45 maintainsthe activity of Lck and Fyn in vivo through dephosphorylation of thesenegative regulatory tyrosines and that this is an important mechanismfor maintaining lymphocyte homeostasis.

A second PTPase which is now believed to play an important positive rolein signal transduction is the intracellular, SH2-domain-containing SHP2(which has also been called SHPTP-2, SHPTP-3, syp, PTP2c, and PTP-1DAdachi, et al., supra!). See generally Saltiel, Am. J Physiol.270:E375-385 (1996); Draznin, Endocrinology 137:2647-2648. SHP2associates, via its SH2 domains, with the receptor for platelet-derivedgrowth factor (PDGF-R), the receptor for epidermal growth factor(EGF-R), with the insulin receptor, and with the predominant substrateof the InsR, insulin receptor substrate 1 (IRS1). Bennett, et al., Proc.Natl. Acad. Sci. 91:7335-7339 (1994); Case, et al., J. Biol. Chem.269:10467-10474 (1994); Kharitonenkov, et al., J. Biol. Chem.270:29189-29193 (1995); Kuhne, et al., J. Biol. Chem. 268:11479-11481(1993). SHP2PTPase activity is required for cellular response to EGF andinsulin, as competitive expression of inactive forms of SHP2 results indiminished signaling events and reduced cellular responses to EGF andinsulin. Milarski and Saltiel, J. Biol. Chem. 269:21239-21243 (1994);Xiao et al., J. Biol. Chem. 269:21244-21248 (1994); Yamauchi et al.,Proc. Natl. Acad. Sci. 92:664-668 (1995). The relevant substrate(s) forthe PTPase domain of SHP2 is not known.

Due to the fundamental role that PTPases play in normal and neoplasticcellular growth and proliferation, a need exists in the art for agentscapable of modulating PTPase activity. On a fundamental level, suchagents are useful for elucidating the precise role of protein tyrosinephosphatases and kinases in cellular signalling pathways and cellulargrowth and proliferation. See generally MacKintosh and MacKintosh, TIBS,19:444-448 (1994).

More importantly, modulation of PTPase activity has important clinicalsignificance. For example, PTP-1B overexpression has been correlatedwith breast and ovarian cancers Weiner et al., J. Natl. Cancer Inst.,86:372-8 (1994); Weiner et al., Am J. Obstet. Gynecol., 170:1177-883(1994)!, and thus agents which modulate PTP-1B activity would be helpfulin elucidating the role of PTP-1B in these conditions and for thedevelopment of effective therapeutics against these disease states. Theimportant role of CD45 in hematopoietic development and T lymphocytefunction likewise indicates a therapeutic utility for PTPase inhibitorsin conditions that are associated with autoimmune disease, and as aprophylaxis for transplant rejection. The antibiotic suramin, which alsoappears to possess anti-neoplastic indications, has recently been shownto be a potent, irreversible, non-competitive inhibitor of CD45. SeeGhosh and Miller, Biochem. Biophys. Res. Comm. 194:36-44 (1993). Thenegative regulatory effects of several PTPases on signaling throughreceptors for growth factors and cytokines, which are implicated innormal cell processing as well as disease states such as cancer andatherosclerosis, also indicate a therapeutic potential for PTPaseinhibitors in diseases of hematopoietic origin.

The PTPase Yop2b is an essential virulence determinant in the pathogenicbacterium Yersinia, responsible for bubonic plague. Bliska et al., Proc.Natl. Acad Sci. USA, 88:1187-91 (1991), and thus an antimicrobialindication exists for PTPase inhibitor compounds, as well.

PTPases have been implicated in diabetic conditions. Experiments withone family of PTPase inhibitors, vanadium derivatives, indicate atherapeutic utility for such compounds as oral adjuvants or asalternatives to insulin for the treatment of hyperglycemia. See Posneret al., J. Biol. Chem., 269:4596-4604 (1994). However, suchmetal-containing PTPase inhibitors act in a fairly non-specific fashionand act with similar potencies against all PTPase enzymes.

In addition to vanadium derivatives, certain organic phosphotyrosinemimetics are reportedly capable of competitively inhibiting PTPasemolecules when such mimetics are incorporated into polypeptideartificial PTPase substrates of 6-11 amino acid residues. For example, a"natural" (phosphorylated tyrosine) PTPase substrate, which may bedepicted by the Formula: ##STR2## has been mimicked by eleven-meroligopeptides containing phosphonomethyl phenylalanine (Pmp), asdepicted bythe schematic Formula: ##STR3## See Chatterjee et al.,"Phosphopeptide substrates and phosphonopeptide inhibitors of proteintyrosine phosphatases," in Peptides. Chemistry and Biology (Rivier andSmith, Eds.), 1992, Escom Science Publishers: Leiden, Netherlands, pp.553-55; Burke et al., Biochemistry, 33:6490-94 (1994). More recently,Burke et al., Biochem. Biophys. Res. Comm. 204(1):129-134 (1994)reported that a particular hexameric peptide sequence comprising a Pmpmoiety or, more preferably, a phosphonodifluoromethyl phenylalanine (F₂Pmp) moiety, as depicted by the schematic Formula: ##STR4##competitively inhibited PTP-1B. However, such hexapeptide inhibitorsnonetheless possess drawbacks for PTPase modulation in vivo. Moreparticularly, the hexapeptide inhibitors described by Burke et al. aresufficiently large and anionic to potentially inhibit efficientmigration across cell membranes, for interaction with the catalyticdomains of transmembrane and intracellular PTPase enzymes which liewithin a cell membrane. A need exists for small, organic-molecule basedPTPase inhibitors having fewer anionic moieties, to facilitate migrationacross cell membranes.

For all of the foregoing reasons, a need exists in the art for novelcompounds effective for modulating, and especially inhibiting, thephosphatase activity of protein tyrosine phosphatase molecules.

SUMMARY OF THE INVENTION

The invention provides compounds and derivatives thereof useful formodulating, and especially inhibiting, the phosphatase activity of oneor more protein tyrosine phosphatase (PTPase) and/or dual specificityphosphatase enzymes. In one aspect, the present invention relates tocompounds having the general structure shown in Formula (A1):

    Y--X--C(R')═C(R")COOR'"                                (A1)

wherein R', R", R'", X and Y are defined below. The inventions furtherprovides salts, esters, prodrugs, solvates, and the like of thecompounds, and compositions comprising these compounds.

DEFINITIONS

In the specification and claims, the term "derivatives" means: arylacrylic acids with structure depicted in Formula (A1) havingsubstitution (with, e.g., hydrogen, hydroxy, halo, amino, carboxy,nitro, cyano, methoxy, etc.) at one or more atoms of the aryl ring.Moreover, "derivatives" includes compounds of the Formula (A1) havingsubstitution at the alkene carbons with, e.g., an electron withdrawinggroup (e.g., Cl, F, Br, CF₃, phenyl) or an electron donating group(e.g., CH₃, alkoxy).

    Y--X--C(R')═C(R")COOR'"                                (A1)

As used herein, the term "attached" signifies a stable covalent bond,certain preferred points of attachment being apparent to those skilledin the art.

The terms "halogen" or "halo" include fluorine, chlorine, bromine, andiodine.

The term "alkyl" includes C₁ -C₁₁ straight chain saturated and C₂ -C₁₁unsaturated aliphatic hydrocarbon groups, C₁ -C₁₁ branched saturated andC₂ -C₁₁ unsaturated aliphatic hydrocarbon groups, C₃ -C₈ cyclicsaturated and C₅ -C₈ unsaturated aliphatic hydrocarbon groups, and C₁-C₁₁ straight chain or branched saturated and C₂ -C₁₁ straight chain orbranched unsaturated aliphatic hydrocarbon groups substituted with C₃-C₈ cyclic saturated and unsaturated aliphatic hydrocarbon groups havingthe specified number of carbon atoms. For example, this definition shallinclude but is not limited to methyl (Me), ethyl (Et), propyl (Pr),butyl (Bu), pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,ethenyl, propenyl, butenyl, penentyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, undecenyl, isopropyl (i-Pr), isobutyl (i-Bu),tert-butyl (t-Bu), sec-butyl (s-Bu), isopentyl, neopentyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,methylcyclopropyl, ethylcyclohexenyl, butenylcyclopentyl, and the like.

The term "substituted alkyl" represents an alkyl group as defined abovewherein the substitutents are independently selected from halo, cyano,nitro, trihalomethyl, carbamoyl, C₀₋₁₁ alkyloxy, arylC₁₋₁₁ alkyloxy,C₀₋₁₁ alkylthio, arylC₀₋₁₁ alkylthio, C₀₋₁₁ alkylamino, arylC₀₋₁₁alkylamino, di(arylC₀₋₁₁ alkyl)amino, C₁₋₁₁ alkylcarbonyl, arylC₁₋₁₁alkylcarbonyl, C₁₋₁₁ alkylcarboxy, arylC₀₋₁₁ alkylcarboxy, C₁₋₁₁alkylcarbonylamino, aryl C₁₋₁₁ alkylcarbonylamino, tetrahydrofuryl,morpholinyl, piperazinyl, hydroxypyronyl, --C₀₋₁₁ alkylCOOR₁, --C₀₋₁₁alkylCONR₂ R₃ wherein R₁, R₂ and R₃ are independently selected fromhydrogen, C₁₋₁₁ alkyl, arylC₀₋₁₁ alkyl, or R₂ and R₃ are taken togetherwith the nitrogen to which they are attached forming a cyclic systemcontaining 3 to 8 carbon atoms with at least one C₁₋₁₁ alkyl, arylC₀₋₁₁alkyl substituent.

The term "alkyloxy" (e.g. methoxy, ethoxy, propyloxy, allyloxy,cyclohexyloxy) represents an alkyl group as defined above having theindicated number of carbon atoms attached through an oxygen bridge. Theterm "alkyloxyalkyl" represents an alkyloxy group attached through analkyl group as defined above having the indicated number of carbonatoms.

The term "alkylthio" (e.g. methylthio, ethylthio, propylthio,cyclohexenylthio and the like) represents an alkyl group as definedabove having the indicated number of carbon atoms attached through asulfur bridge. The term "alkylthioalkyl" represents an alkylthio groupattached through an alkyl group as defined above having the indicatednumber of carbon atoms.

The term "alkylamino" (e.g. methylamino, diethylamino, butylamino,N-propyl-N-hexylamino, (2-cyclopentyl)propylamino, hexenylamino,pyrrolidinyl, piperidinyl and the like) represents one or two alkylgroups as defined above having the indicated number of carbon atomsattached through an amine bridge. The two alkyl groups maybe takentogether with the nitrogen to which they are attached forming a cyclicsystem containing 3 to 11 carbon atoms with at least one C₁₋₁₁ alkyl,arylC₀₋₁₁ alkyl substituent. The term "alkylaminoalkyl" represents analkylamino group attached through an alkyl group as defined above havingthe indicated number of carbon atoms.

The term "alkylcarbonyl" (e.g. cyclooctylcarbonyl, pentylcarbonyl,3-hexenylcarbonyl) represents an alkyl group as defined above having theindicated number of carbon atoms attached through a carbonyl group. Theterm "alkylcarbonylalkyl" represents an alkylcarbonyl group attachedthrough an alkyl group as defined above having the indicated number ofcarbon atoms.

The term "alkylcarboxy" (e.g. heptylcarboxy, cyclopropylcarboxy,3-pentenylcarboxy) represents an alkylcarbonyl group as defined abovewherein the carbonyl is in turn attached through an oxygen. The term"alkylcarboxyalkyl" represents an alkylcarboxy group attached through analkyl group as defined above having the indicated number of carbonatoms.

The term "alkylcarbonylamino" (e.g. hexylcarbonylamino,cyclopentylcarbonyl-aminomethyl, methylcarbonylaminophenyl) representsan alkylcarbonyl group as defined above wherein the carbonyl is in turnattached through the nitrogen atom of an amino group. The nitrogen groupmay itself be substituted with an alkyl or aryl group. The term"alkylcarbonylaminoalkyl" represents an alkylcarbonylamino groupattached through an alkyl group as defined above having the indicatednumber of carbon atoms. The nitrogen group may itself be substitutedwith an alkyl or aryl group.

The term "aryl" represents an unsubstituted, mono-, di- ortrisubstituted monocyclic, polycyclic, biaryl and heterocyclic aromaticgroups covalently attached at any ring position capable of forming astable covalent bond, certain preferred points of attachment beingapparent to those skilled in the art (e.g., 3-indolyl, 4-imidazolyl).The aryl substituents are independently selected from the groupconsisting of halo, nitro, cyano, trihalomethyl, hydroxypyronyl, C₁₋₁₁alkyl, arylC₁₋₁₁ alkyl, C₀₋₁₁ alkyloxyC₀₋₁₁ alkyl, arylC₁₋₁₁alkyloxyC₀₋₁₁ alkyl, C₀₋₁₁ alkylthioC₀₋₁₁ alkyl, arylC₀₋₁₁alkylthioC₀₋₁₁ alkyl, C₀₋₁₁ alkylaminoC₀₋₁₁ alkyl, arylC₀₋₁₁alkylaminoC₀₋₁₁ alkyl, di(arylC₁₋₁₁ alkyl)aminoC₀₋₁₁ alkyl, C₁₋₁₁alkylcarbonylC₀₋₁₁ alkyl, arylC₀₋₁₁ alkylcarbonylC₀₋₁₁ alkyl, C₁₋₁₁alkylcarboxyC₀₋₁₁ alkyl, arylC₁₋₁₁ alkylcarboxyC₀₋₁₁ alkyl, C₁₋₁₁alkylcarbonylaminoC₀₋₁₁ alkyl, arylC₁₋₁₁ alkylcarbonylaminoC₀₋₁₁ alkyl,--C₀₋₁₁ alkylCOOR₄, --C₀₋₁₁ alkylCONR₅ R₆ wherein R₄, R₅ and R₆ areindependently selected from hydrogen, C₁ -C₁₁ alkyl, arylC₀ -C₁₁ alkyl,or R₅ and R₆ are taken together with the nitrogen to which they areattached forming a cyclic system containing 3 to 8 carbon atoms with atleast one C₁ -C₁₁ alkyl, arylC₀ -C₁₁ alkyl substituent.

The definition of aryl includes but is not limited to phenyl, biphenyl,naphthyl, dihydronaphthyl, tetrahydronaphthyl, indenyl, indanyl,azulenyl, anthryl, phenanthryl, fluorenyl, pyrenyl, thienyl,benzothienyl, isobenzothienyl, 2,3-dihydrobenzothienyl, furyl, pyranyl,benzofuranyl, isobenzofuranyl, 2,3-dihydrobenzofuranyl, pyrrolyl,indolyl, isoindolyl, indolizinyl, indazolyl, imidazolyl, benzimidazolyl,pyridyl, pyrazinyl, pyradazinyl, pyrimidinyl, triazinyl, quinolyl,isoquinolyl, 4H-quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, chromanyl, benzodioxolyl,piperonyl, purinyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl,isothiazolyl, benzthiazolyl, oxazolyl, isoxazolyl, benzoxazolyl,oxadiazolyl, thiadiazolyl.

The term "arylalkyl" (e.g. (4-hydroxyphenyl)ethyl,(2-aminonaphthyl)hexenyl, pyridylcyclopentyl) represents an aryl groupas defined above attached through an alkyl group as defined above havingthe indicated number of carbon atoms.

The term "arylcarbonyl" (e.g. 2-thiophenylcarbonyl,3-methoxyanthrylcarbonyl, oxazolylcarbonyl) represents an aryl group asdefined above attached through a carbonyl group.

The term "arylalkylcarbonyl" (e.g. (2,3-dimethoxyphenyl)propylcarbonyl,(2-chloronaphthyl)pentenylcarbonyl, imidazolylcyclopentylcarbonyl)represents an arylalkyl group as defined above wherein the alkyl groupis in turn attached through a carbonyl.

The term "signal transduction" is a collective term used to define allcellular processes that follow the activation of a given cell or tissue.Examples of signal transduction include but are not in any way limitedto cellular events that are induced by polypeptide hormones and growthfactors (e.g. insulin, insulin-like growth factors I and II, growthhormone, epidermal growth factor, platelet-derived growth factor),cytokines (e.g. interleukines), extracellular matrix components, andcell-cell interactions.

Phosphotyrosine recognition units/tyrosine phosphate recognitionunits/phosphotyrosine recognition units are defined as areas or domainsof proteins or glycoproteins that have affinity for molecules containingphosphorylated tyrosine residues (pTyr). Examples of pTyr recognitionunits include but are not in any way limited to: PTPases, SH2 domainsand PTB domains.

PTPases are defined as enzymes with the capacity to dephosphorylatepTyr-containing proteins or glycoproteins. Examples of PTPases includebut are not in any way limited to: intracellular PTPases (e.g. PTP-1B,TC-PTP, PTP-1C, PTP-1D,PTP-D1, PTP-D2), receptor-type PTPases (e.g.PTPα, PTPε, PTPβ, PTPγ, CD45, PTPκ, PTPμ), dual specificity phosphatases(e.g. VH1, VHR, cdc25) and other PTPases such as LAR, SHP-1, SHP-2,PTP-1H, PTPMEGI, PTP-PEST, PTPζ, PTPS31, IA-2 and HePTP and the like.

Modulation of cellular processes is defined as the capacity of compoundsof the invention to 1) either increase or decrease ongoing, normal orabnormal, signal transduction, 2) initiate normal signal transduction,and 3) initiate abnormal signal transduction.

Modulation of pTyr-mediated signal transduction/modulation of theactivity of molecules with pTyr recognition units is defined as thecapacity of compounds of the invention to 1) increase or decrease theactivity of proteins or glycoproteins with pTyr recognition units (e.g.PTPases, SH2 domains or PTB domains) or to 2) decrease or increase theassociation of a pTyr-containing molecule with a protein or glycoproteinwith pTyr recognition units either via a direct action on the pTyrrecognition site or via an indirect mechanism. Examples of modulation ofpTyr-mediated signal transduction/modulation of the activity ofmolecules with pTyr recognition units, which are not intended in any waylimiting to the scope of the invention claimed, are: a) inhibition ofPTPase activity leading to either increased or decreased signaltransduction of ongoing cellular processes; b) inhibition of PTPaseactivity leading to initiation of normal or abnormal cellular activity;c) stimulation of PTPase activity leading to either increased ordecreased signal transduction of ongoing cellular processes; d)stimulation of PTPase activity leading to initiation of normal orabnormal cellular activity; e) inhibition of binding of SH2 domains orPTB domains to proteins or glycoproteins with pTyr leading to increaseor decrease of ongoing cellular processes; f) inhibition of binding ofSH2 domains or PTB domains to proteins or glycoproteins with pTyrleading to initiation of normal or abnormal cellular activity.

A subject is defined as any mammalian species, including humans.

DETAILED DESCRIPTION

This application relates to compounds having the general structure shownin Formula (A1):

    Y--X--C(R')═C(R")COOR'"                                (A1)

wherein

(i) R' and R" are independently selected from the group consisting ofhydrogen, halo, cyano, nitro, trihalomethyl, alkyl, arylalkyl,

(ii) R'" is selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, arylalkyl

(iii) X is aryl,

(iv) Y is selected from hydrogen or ##STR5## wherein (*) indicates apotential point of attachment to X and all other positions aresubstituted as described below.

(1) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A2): ##STR6## wherein at least one of R₁, R₂ and R₃substituents has the general structure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ and R₃ are independently selected fromthe group consisting of hydrogen, alkyl, substituted alkyl, aryl,arylalkyl.

(2) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A3): ##STR7##

wherein at least one of R₁, R₂ and R₃ substituents has the generalstructure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ and R₃ are independently selected fromthe group consisting of: hydrogen, alkyl, substituted alkyl,alkylcarbonyl, substituted alkylcarbonyl, aryl, arylalkyl, arylcarbonyl,arylalkylcarbonyl.

(3) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A4): ##STR8## wherein at least one of R₁, R₂ substituentshas the general structure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" X are defined as above in Formula (A1), and whereinthe remaining of R₁, R₂ is defined as above in Formula (A2).

(4) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A5): ##STR9## wherein at least one of R₁ and R₂substituents has the general structure depicted in Formula (B) ##STR10##wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁ and R₂ is defined as above in Formula (A2).

(5) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A6): ##STR11## wherein at least one of R₁, R₂, R₃ and R₄substituents has the general structure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂, R₃ and R₄ have the same definition asR₁, R₂ and R₃ in Formula (A2), with the proviso that when R₃ and R₄ areselected from substituted phenyl or substituted furyl then the phenyland furyl substituents exclude hydroxy, halo, trifluoromethyl, C₁₋₆alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, amino, C₁₋₆ alkylamino, di(C₁₋₆alkyl)amino, phenylC₁₋₆ alkylamino and di(phenylC₁₋₆ alkyl)amino.

(6) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A6): ##STR12## wherein R₄ is selected from --COR₅, --COOR₆,--CONR₇ R₈ wherein R₅ thru R₈ are selected from hydrogen, alkyl,substituted alkyl, aryl, arylalkyl, or R₇ and R₈ are taken together withthe nitrogen to which they are attached forming a cyclic systemcontaining 3 to 8 carbon atoms with at least one alkyl, aryl, arylalkylsubstituent, and wherein at least one of R₁, R₂, and R₃ substituents hasthe general structure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ and R₃ are defined as above in Formula(A2).

(7) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A6): ##STR13## wherein R₁, R₂, R₃ and R₄ are defined asabove in (6).

(8) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A7): ##STR14## wherein R₂ is selected from --COR₅, --COOR₆,--CONR₇ R₈ wherein R₅ thru R₈ are defined as above in (6) and wherein atleast one of R₁ and R₃ substituents has the general structure depictedin Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁ and R₃ are defined as above in Formula (A2).

(9) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A8): ##STR15## wherein at least one of R₁ and R₂substituents has the general structure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁ and R₂ is defined as above in Formula (A2),and wherein m is an integer between 0 and 4 and each R₃ is independentlyselected from the group consisting of halo, nitro, cyano, trihalomethyl,hydroxypyronyl, alkyl, arylalkyl, C₀₋₁₁ alkyloxyC₀₋₁₁ alkyl, arylC₀₋₁₁alkyloxyC₀₋₁₁ alkyl, C₀₋₁₁ alkylthioC₀₋₁₁ alkyl, arylC₀₋₁₁alkylthioC₀₋₁₁ alkyl, C₀₋₁₁ alkylaminoC₀₋₁₁ alkyl, arylC₀₋₁₁alkylaminoC₀₋₁₁ alkyl, di(arylC₁₋₁₁ alkyl)aminoC₀₋₁₁ alkyl, C₁₋₁₁alkylcarbonylC₀₋₁₁ alkyl, arylC₁₋₁₁ alkylcarbonylC₀₋₁₁ alkyl, C₁₋₁₁alkylcarboxyC₀₋₁₁ alkyl, arylC₁₋₁₁ alkylcarboxyC₀₋₁₁ alkyl, C₁₋₁₁alkylcarbonylaminoC₀₋₁₁ alkyl, arylC₁₋₁₁ alkylcarbonylaminoC₀₋₁₁ alkyl,--C₀₋₁₁ alkylCOOR₄, --C₀₋₁₁ alkylCONR₅ R₆ wherein R₄, R₅ and R₆ areindependently selected from hydrogen, C₁ -C₁₁ alkyl, arylC₀ -C₁₁ alkyl,or R₅ and R₆ are taken together with the nitrogen to which they areattached forming a cyclic system containing 3 to 8 carbon atoms with atleast one C₁ -C₁₁ alkyl, arylC₀ -C₁₁ alkyl substituent.

(10) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A8): ##STR16## wherein R₁ is selected from --COR₅, --COOR₆,--CONR₇ R₈ wherein R₅ thru R₈ are defined as above in (6) and wherein R₂has the general structure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein m is an integer between 0 and 4 and each R₃ is defined as abovein (9).

(11) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A9): ##STR17## wherein m is an integer between 0 and 3 andwherein R₁, R₂ each R₃ is defined as above in (9).

(12) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A9): ##STR18## wherein either R₁ or R₂ is selected from--COR₅, --COOR₆, --CONR₇ R₈ wherein R₅ thru R₈ are defined as in (6) andwherein the remainder of R₁ and R₂ is defined as above in (9), andwherein m is an integer between 0 and 3 and each R₃ is defined as abovein (9).

(13) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A 10): ##STR19## wherein Z₁ and Z₂ are independentlyselected from the group consisting of OR₃, SR₄, NR₅ R₆ and wherein atleast one of R₁, R₂ substituents has the general structure depicted inFormula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ is defined as above in Formula (A2), andwherein R₃, R₄, R₅, R₆ are independently selected from hydrogen, alkyl,substituted alkyl, alkylcarbonyl, substituted alkylcarbonyl, aryl,arylalkyl, arylcarbonyl, arylalkylcarbonyl.

(14) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A11): ##STR20## wherein at least one of R₁, R₂, and R₃substituents has the general structure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein R', R", R'" and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ and R₃ are defined as above in Formula(A2).

Preferred compositions of the invention include compositions comprisingcompounds as defined above in structural formula (A1), (A2), (A3), (A4),(A5), (A6), (A7), (A8), (A9), (A10), (A11) (or pharmaceuticallyacceptable salts, prodrugs, esters, or solvates of these compounds) inadmixture with a pharmaceutically acceptable diluent, adjuvent, orcarrier.

Provided according to the invention, therefore, are novel compoundswhich modulate the activity of PTPase or other molecules with pTyrrecognition unit(s) as well as previously known aryl acrylic acidcompounds which modulate the activity of PTPase or other molecules withpTyr recognition unit(s).

Another aspect of the present invention provides compositions comprisingPTPase modulating compounds of the invention suitable for administrationto a mammalian host.

In a preferred embodiment the compounds of the invention act asinhibitors of PTPases, e.g. protein tyrosine phosphatases involved inthe regulation of tyrosine kinase signaling pathways. Preferredembodiments include modulation of receptor-tyrosine kinase signalingpathways via interaction with regulatory PTPases, e.g. the signalingpathways of the insulin receptor, the IGF-I receptor and other membersof the insulin receptor family, the EGF-receptor family, theplatelet-derived growth factor family, the nerve growth factor receptorfamily, the hepatocyte growth factor receptor family, the growth hormonereceptor family and members of other receptor-type tyrosine kinasefamilies. Further preferred embodiments of the invention is modulationof non-receptor tyrosine kinase signaling through modulation ofregulatory PTPases, e.g. modulation of members of the Src kinase family.One type of preferred embodiments of the invention relates to modulationof the activity of PTPases that negatively regulate signal transductionpathways. Another type of preferred embodiments of the inventions relateto modulation of the activity of PTPases that positively regulate signaltransduction pathways.

In a preferred embodiment compounds of the inventions act as modulatorsof the active site of PTPases. In another preferred embodiment thecompounds of the invention modulate the activity of PTPases viainteraction with structures positioned outside the active sites of theenzymes, preferably SH2 domains. Further preferred embodiments includemodulation of signal transduction pathways via binding of the compoundsof the invention to SH2 domains or PTB domains of non-PTPase signalingmolecules.

Other preferred embodiments include use of the compounds of theinvention for modulation of cell-cell interactions as well ascell-matrix interactions.

As a preferred embodiment, the compounds of the invention may be used astherapeutics to inhibit PTPases involved in the regulation of theinsulin receptor tyrosine kinase signaling pathway in patients with typeI diabetes, type II diabetes, impaired glucose tolerance, insulineresistance and obesity. Further preferred embodiments include use of thecompounds of the invention for treatment of disorders with general orspecific dysfunction of PTPase activity, e.g. proliferative disordersincluding neoplastic diseases and psoriosis. As an other embodiment, thecompounds of the invention may be used in pharmaceutical preparationsfor treatment of osteoporosis.

Preferred embodiments of the invention further include use of compoundsof the invention in pharmaceutical preparations to increase thesecretion or action of growth hormone and its analogs or somatomedinsincluding IGf-I and IGF-2 by modulating the activity of PTPases or othersignal transduction molecules with affinity for phosphotyrosine involvedcontrolling or inducing the action of these hormones or any regulatingmolecule.

To those skilled in the art, it is well known that the current andpotential uses of growth hormone in humans are varied and muti-tudinous.Thus, compounds of the invention can be administered for purposes ofstimulating the release of growth hormone from the pituitary or increaseits action on target tissues thereby leading to similar effects or usesas growth hormone itself. The uses of growth hormone maybe summarized asfollows: stimulation of growth hormone release in the elderly;prevention of catabolic side effects of glucocorticoids; treatment ofosteoporosis, stimulation of the immune system; treatment ofretardation, accelaration of wound healing; accelerating bone fracturerepair; treatment of growth retardation; treating renal failure orinsufficiency resulting in growth retardation; treatment ofphysiological short stature including growth hormone deficient childrenand short stature associated with chronic illness; treatment of obesityand growth retardation associated with obesity; treating growthretardation associated with the Pader-Willi syndrom and Turner'ssyndrom; accelerating the recovery and reducing hospitalization of burnpatients; treatment of intrauterine growth retardation, skeletaldysplasia, hypercortisolism and Cushings syndrome; induction ofpulsatile growth hormone release; replacement of growth hormone instressed patients; treatment of osteochondro-dysplasis, Noonanssyndrome, schizophrenia, depressions, Alzheimer's disease, delayed woundhealing and psychosocial deprivation; treatment of pulmonary dysfunctionand ventilator dependency; attenuation of protein catabolic responsesafter major surgery; reducing cachexia and protein loss due to chronicillness such as cancer or AIDS; treatment of hyperinsulinemia includingnesidio-blastosis; adjuvant treatment for ovulation induction;stimulation of thymic development and prevention of age related declineor thymic function; treatment of immunosuppresed patients; improvementin muscle strength, mobility, maintenance of skin thickness, metabolichomeostasis, renal homeostasis in the frail elderly; stimulation ofosteoblasts, bone remodelling and cartilage growth; stimulation of theimmune system in companion animals and treatment of disorder of aging incompanion animals; growth promotant in livestock and stimulation of woolgrowth in sheep.

The compounds of the invention may be used in pharmaceuticalpreparations for treatment of various disorders of the immune system,either as stimulant or suppressor of normal or perturbed immunefunctions, including autoimmune reactions. Further embodiments of theinvention for treatment of allergic reactions, e.g. asthma, dermalreactions, conjunctivitis.

In another embodiment, compounds of the invention may be used inpharmaceutical preparations for prevention or induction of plateletaggregation.

In yet another embodiment, compounds of the invention may be used inpharmaceutical preparations for treatment of infectious disorders. Inparticular, the compounds of the invention may be used for treatment ofinfectious disorders caused by Yersinia and other bacteria as well asdisorders caused by viruses or other micro-organisms.

Compounds of the invention may additionally be used for treatment orprevention of diseases in animals, including commercially importantanimals.

Also included in the present invention is a process for isolation ofPTPases via affinity purification procedures based on the use ofimmobilized compounds of the invention using procedures well-known tothose skilled in the art.

The invention is further directed to a method for detecting the presenceof PTPases in cell or in a subject comprising

(a) contacting said cell or an extract thereof with labeled compounds ofthe invention.

(b) detecting the binding of the compounds of the invention or measuringthe quantity bound, thereby detecting the presence or measuring thequantity of certain PTPases.

The invention further relates to analysis and identification of thespecific functions of certain PTPases by modulating their activity byusing compounds of the invention in cellular assay systems or in wholeanimals.

The invention further provides methods for making compounds (A1), (A2),(A3), (A4), (A5), (A6), (A7), (A8), (A9), (A 10), (A11) of the presentinvention having PTPase-modulatory/inhibitory activity. In preferredmethods, compounds of the invention are synthesized in a multi-componentcombinatorial array, which permits rapid synthesis of numerous,structurally related compounds for subsequent evaluation. In preferredsynthesis protocols, the acrylic acid moiety of a compound is protectedduring synthesis by, e.g., esterification with a tert-butyl protectinggroup. Thus, a preferred method of making compounds of the inventioncomprises use of a protected acrylic acid reagent and removal of theprotective group by, e.g., treatment of a precursor ester compound withacid. Optionally, such a method includes further esterifying orsalifying the acrylic acid product thereby obtained.

The compounds of formula (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8),(A9), (A 10), (A 11) may be prepared by procedures known to thoseskilled in the art from known compounds or readily preparableintermediates. General synthetic procedures and examples are as follow:

General method for the removal of tert-butyl esters ##STR21##

Unless otherwise stated, tert-butyl esters were converted to theircorresponding carboxylic acids via treatment with a solution of 50%trifluoroacetic acid in dichloromethane for 1 hour at 23° C. The solventwas removed in vacuo and the residue was azeotroped with toluene oracetonitrile to yield the corresponding carboxylic acid.

General method for the synthesis of compounds (A1) and (A5)

Method 1 ##STR22##

By allowing a compound of formula (1) wherein LG is a suitable leavinggroup such as bromo, iodo, or triflate to react with compound of formula(2) wherein Z is hydrogen (Heck reaction: J. Org. Chem., 1977, 42,3903), or trialkyltin (Stille reaction: J. Am. Chem. Soc., 1991, 113,9585), or B(OH)₂ (Suzuki reaction: J. Am. Chem. Soc., 1989, 111, 314)and wherein R', R", R'" and X are defined as above for formula (A1).

These reactions may be carried out neat or in a solvent such asdimethylformamide (DMF), tetrahydrofuran (THF), or toluene, in thepresence of a catalyst (e.g. Pd(OAc)₂, Pd(PPh₃)₄, Pd₂ dba₃), a ligand(e.g. Ph₃ P, Ph₃ As, (o-tolyl)₃ P) and a base (e.g. K₂ CO₃, CsCO₃, Et₃N) at temperatures ranging from 23° C. to 130° C., for 1 to 60 hours.

EXAMPLES ##STR23##

Prepared according to Patel et al (J. Org. Chem., 1977, 42, 3903).

¹ H NMR of 3 (400 MHz, CDCl₃) δ1.5 (s, 9H), 6.4 (d, 1H), 7.6 (m, 3H),8.05 (d, 2H). ##STR24##

Prepared according to Patel et al (J. Org. Chem., 1977, 42, 3903).

¹ H NMR of 4 (400 MHz, CDCl₃) δ1.5 (s, 9H), 6.4 (d, 1H), 7.55 (d, 1H),7.6 (d, 2H), 7.8 (d, 2H), 9.95 (s, 1H). ##STR25##

Prepared according to Patel et al (J. Org. Chem., 1977, 42, 3903).

¹ H NMR of 5 (400 MHz, CDCl₃) δ1.44 (s, 9H), 6.26 (d, 1H), 7.18 (d, 1H),7.56 (d, 1H), 7.74 (d, 1H). ##STR26##

Prepared according to Patel et al (J. Org. Chem. 1977, 42, 3903).

¹ H NMR of 6 (400 MHz, CDCl₃) δ1.5 (s, 18H), 6.42 (d, 2H), 7.6 (m, 6H),7.9 (d, 4H). ##STR27##

Prepared according to Patel et al (J. Org. Chem. 1977, 42, 3903).

¹ H NMR of 7 (400 MHz, CDCl₃) δ1.5 (s, 18H), 6.2 (d, 2H), 7.1 (d, 2H),7.35 (d, 2H), 7.5 (s, 2H), 7.7 (d, 2H). ##STR28##

To 11 g of 4,4'-dibromobenzil (30 mmol, 1.0 equiv), 67 mg of palladium(II) acetate (0.3 mmol, 0.01 equiv), 365 mg of tri-o-tolylphosphine (1.2mmol, 0.04 equiv) was added 200 mL of dimethylformamide followed by 4.2mL (30 mmol, 1.0 equiv) of triethylamine. The mixture was placed in a100° C. preheated bath and 4.4 mL of tert-butylacrylate (30 mmol, 1.0equiv) in 30 mL of dimethylforamide was added dropwise over 1 hour. Thereaction mixture was heated at 100° C. for 12 hours, cooled to 23° C.and the solvent was removed in vacuo. Ethyl acetate was added and theorganic layer was washed with water and dried over sodium sulfate. Thesolvent was removed and the residue (mixture of dibromobenzil, mono andbis-tert-butylacrylate benzil) was recrystallized from hot 30%dichloromethane in hexane. The solid which crashed out (mixture ofdibromobenzil and mono-tert-butylacrylate benzil) was filtered off andtreated with 20% trifluoroacetic acid in dichloromethane. After 20minutes, the mono-tert-butylacrylate benzil 8 was filtered off andwashed with 20% trifluoroacetic acid in dichloromethane (1.4 gisolated). The mother liquor (mixture of mono and bis-tert-butylacrylatebenzil) was recovered and purified by flash chromatography (ethylacetate-hexane eluant) to yield 2.4 g of the mono-tert-butylacrylatedione which was treated with 20% trifluoroacetic acid in dichloromethaneto give 2.2 g of 8. The combined total yield of 8 was 3.6 g (34%). ¹ HNMR of 8 (400 MHz, d₆ -DMSO) δ6.7 (d, 1H), 7.6 (d, 1H), 7.8 (s, 4H), 7.9(s, 4H). ##STR29##

¹ H NMR of 9 (400 MHz, d₆ -DMSO) δ6.7 (d, 2H), 7.6 (d, 2H), 7.9 (s, 8H).##STR30##

Prepared according to Patel et al (J. Org. Chem., 1977, 42, 3903).

¹ H NMR of 10 (400 MHz, CDCl₃ --CD₃ OD 9:1) δ1.45 (s, 9H), 6.42 (d, 1H),6.5 (d, 1H), 7.55 (d, 1H), 7.6 (dd, 4H), 7.68 (d, 1H), 7.92 (dd, 4H).##STR31##

To a solution of 10 (1 equiv) in dichloromethane was added octylamine (1equiv), EDCI (1.3 equiv) and 4-dimethylaminopyridine (0.5 equiv) at 23°C. The solution was stirred overnight, diluted with ethyl acetate,washed with 1N HCl and saturated sodium bicarbonate and dried oversodium sulfate. The residue was purified by flash chromatography (ethylacetate-hexane eluant) and the solvent was removed in vacuo to yieldcompound 11. ¹ H NMR of 11 (400 MHz, CDCl₃) δ0.9 (t, 3H), 1.25 (s br,10H), 1.5 (s, 9H), 1.55 (s br, 2H), 3.35 (dd, H), 5.6 (t br, 1H), 6.449d, 1H), 6.48 (d, 1H), 7.58 (m, 6H), 7.92 (d, 4H). ##STR32##

Same procedure as compound 11. ¹ H NMR of 12 (400 MHz, CDCl₃) δ1.5 (s,9H), 2.83 (t, 2H), 3.62 (dt, 2H), 5.82 (t br, 1H), 6.4 (m, 2H), 7.18 (m,5H), 7.6 (m, 6H), 7.9 (m, 4H). ##STR33## Method 2 ##STR34##

By allowing a compound of formula (1) as defined above to react withpolymer bound compound of formula (14) wherein Z, R' and R" are definedas above in method 1.

These reactions may be carried out on functionalized cross linkedpolystyrene polymers such as Merrifield resin, Wang resin, Rink resin,"TENTAGEL™" which is a polyethylene-polystyrene polymer resin, in asolvent such as dimethylformamide (DMF), tetrahydrofuran (THF), ortoluene, in the presence of a catalyst (e.g. Pd(OAc)₂, Pd(PPh₃)₄, Pd₂dba₃), a ligand (e.g. Ph₃ P, Ph₃ As, (o-tolyl)₃ P) and a base (e.g. K₂CO₃, CsCO₃, Et₃ N) at temperatures ranging from 23° C. to 130° C., for 1to 60 hours.

EXAMPLES ##STR35##

For leading references see: a) Mathias (Synthesis 1979, 561). b)Sarantakis et al (Biochem. Biophys. Res. Commun. 1976, 73, 336). c)Hudson et al (Peptide Chemistry 1985 (Kiso, Y., ed.), 1986, ProteinResearch Foundation, Osaka.). d) Wang (J. Am. Chem. Soc. 1973, 95,1328). e) Lu et al (J. Org. Chem. 1981, 46, 3433.) e) Morphy et al(Tetrahedron Letters 1996, 37, 3209). e) Yedidia et al (Can. J. Chem.1980, 58, 1144).

To 10 g (11.2 mmol, 1 equiv) of Wang resin in 80 mL of drydichloromethane was added 33.6 mmol (3 equiv) of diisopropylcarbodiimideand the mixture was sonnicated under N₂ for 2 hours (final bathtemperature was 40° C.). Freshly distilled acrylic acid (33.6 mmol, 3equiv) and 4-dimethylaminopyridine (11.2 mmol, 1 equiv) were added andthe mixture was magnetically stirred for 16 hours at ambienttemperature. The resin was filtered and thoroughly washed withdichloromethane (500 mL), methanol (500 mL), dimethylformamide (500 mL),dichloromethane (500 mL) and methanol (500 mL) and dried in vacuo (0.1mmHg) for 24 hours. The coupling was repeated and resin 15 was filtered,washed and dried as above, and used directly in the next step. ##STR36##

To 8.2 g of acrylate Wang resin 15 was added 10.4 g (28.3 mmol) of4,4'-dibromobenzil, 437 mg of palladium (II) acetate (1.95 mmol), 1.25 gof tri-o-tolylphosphine (4.11 mmol), 95 mL of dimethylformamide followedby 3.3 mL (23.7 mmol) of triethylamine. The mixture was placed in a 100°C. preheated bath and stirred magnetically at 200 rpm for 2 hours. Theresin was filtered hot and washed thoroughly with hot dimethylformamide(500 mL), hot acetic acid (500 mL), methanol (500 mL), dichloromethane(500 mL), dimethylformamide (500 mL), dichloromethane (500 mL) andmethanol (500 mL) and dried in vacuo (0.1 mmHg) for 24 hours. The linkerwas cleaved from the resin with a solution of 20% trifluoroacetic acidin dichloromethane for 20 min at ambient temperature. ¹ H NMR formonobromomonoacid linker (400 MHz, d₆ -DMSO) δ6.7 (d, 2H), 7.6 (d, 2H),7.8 (s, 4H), 7.9 (s, 4H). ##STR37##

To 10.2 g resin 16 was added 5.41 mL (37 mmol) of tert-butylacrylate,132 mg of palladium (II) acetate (0.592 mmol), 0.360 g oftri-o-tolylphosphine (1.18 mmol), 31 mL of dimethylformamide followed by1 mL (7.4 mmol) of triethylamine. The mixture was placed in a 100° C.preheated bath and stirred magnetically at 200 rpm for 18 hours. Theresin was filtered hot and washed thoroughly with hot dimethylformamide(500 mL), hot acetic acid (500 mL), methanol (500 mL), dichloromethane(500 mL), dimethylformamide (500 mL), dichloromethane (500 mL) andmethanol (500 mL) and dried in vacuo (0.1 mmHg) for 24 hours. The linkerwas cleaved from the resin with a solution of 20% trifluoroacetic acidin dichloromethane for 20 min at ambient temperature. ¹ H NMR for diacidlinker (400 MHz, d₆ -DMSO) δ6.7 (d, 2H), 7.6 (d, 2H), 7.9 (s, 8H).##STR38##

To 1 g of acrylate resin 15 was added 1.02 g (2.8 mmol) ofmono-bromo-mono-tert-butylacrylate benzil (8), 0.044 g of palladium (II)acetate (0.19 mmol), 0.130 g of tri-o-tolylphosphine (0.41 mmol), 10 mLof dimethylformamide followed by a solution of 0.76 mL (5.7 mmol) oftriethylamine in 10 mL of dimethylformamide. The mixture was placed in a100° C. preheated bath and stirred magnetically at 200 rpm for 2 hours.The resin was filtered hot and washed thoroughly with hotdimethylformamide (50 mL), water (50 mL), 10% sodium bicarbonate (50mL),10% aqueous acetic acid (50 mL), water (50 mL), methanol (50 mL),dichloromethane (50 mL), methanol (50 mL), dichloromethane (50 mL) anddried in vacuo (0.1 mmHg) for 24 hours. The linker was cleaved from theresin with a solution of 20% trifluoroacetic acid in dichloromethane for20 min at ambient temperature. ¹ H NMR for diacid linker (400 MHz, d₆-DMSO) δ6.7 (d, 2H), 7.6 (d, 2H), 7.9 (s, 8H). ##STR39##

Resin 18 was treated with a 1.0M solution of oxalyl chloride indichloromethane in the presence of a catalytic amount ofdimethylformamide for 1 hour and filtered. The resin was subsequentlytreated with a dichloromethane solution containing the alcohol (ROH),pyridine and 4-dimethylaminopyridine for 20 hours at 23° C. to yield themonoester resin 19. ##STR40##

Resin 18 was treated with a 1.0M solution of oxalyl chloride indichloromethane in the presence of a catalytic amount ofdimethylformamide for 1 hour and filtered. The resin was subsequentlytreated with a dichloromethane solution containing the aromatic amine(ArN(R₁)H), pyridine and 4-dimethylaminopyridine for 20 hours at 23° C.to yield the monoamide resin 20. ##STR41##

Resin 18 was treated with a dichloromethane solution containing theamine (R₁ R₂ NH), EDCI and 4-dimethylaminopyridine for 20 hours at 23°C. to yield the monoamnide resin 21.

General methods for the synthesis of compounds (A2) and (A10)

Method 1 ##STR42##

By allowing an aldehyde (R₁ CHO) wherein R₁ is defined as above informula (A10) to react with itself.

These reactions may be carried out in a solvent or combination ofsolvents such as tetrahydrofuran (THF), dichloromethane (CH₂ Cl₂), inthe presence of a catalyst (e.g. TiCl₃), and a base (e.g. pyridine) attemperatures ranging from -78° C. to 23° C., for 1 to 60 hours.

EXAMPLES ##STR43##

Prepared according to Araneo et al (Tetrahedron Lett. 1994, 35, 2213).The reaction was stirred for 4 hrs at 23° C. ¹ H NMR of 22 (400 MHz,CDCl₃) δ1.55 (s, 18H), 4.65 (s, 2H), 6.27 (d, 2H), 7.05 (d, 4H), 7.31(d, 4H), 7.5 (d, 2H). ##STR44##

¹ H NMR of 23 (400 MHz, CD₃ OD) δ4.65 (s, 2H), 6.4 (d, 2H), 7.15 (d,4H), 7.4 (d, 4H), 7.6 (d, 2H). MS ESI (neg ion) for M-H!⁻ : 353(calculated 354).

Method 2 ##STR45##

By allowing a compound of formula (A 10)-1 prepared as above to reactwith a acid chloride (R₂ CO₂ H) and by subsequently oxidizing (A10)-2wherein R₁ and R₂ are defined as in formula (A2).

The first step in this reaction may be carried out in a solvent such astetrahydrofuran (THF), dichloromethane (CH₂ Cl₂), in the presence ofdiisopropyl carbodiimide (DIC) and a base (e.g. 4-dimethylaminopyridine)at temperatures ranging from 0° C. to 23° C., for 1 to 60 hours. Thesecond step in this reaction may be carried out in a solvent such asdichloromethane (CH₂ Cl₂), in the presence of an oxidizing reagent (e.g.tetrapropylammonium perruthenate (VII) (TPAP)) and activated 4 Åmolecular sieves at temperatures ranging from 0° C. to 23° C., for 1 to60 hours.

EXAMPLES ##STR46##

To 50 mg of diol 22 (1 equiv) in 1 mL of dichloromethane was addeddiisopropyl carbodiimide (0.4 equiv) and the reaction was stirred for 1hour at 23° C. To the solution was added 4-dimethylaminopyridine (0.1equiv) followed by para-methoxybenzoic acid (0.4 equiv) in 5 mL oftetrahydrofuran and the mixture was stirred for an additional 3 hours at23° C. The reaction was diluted with ethyl acetate and washed with 1NHCl, saturated sodium bicarbonate and the organic layer was dried oversodium sulfate. The crude mixture was separated using radialchromatography (ethyl acetate-hexane eluent). ¹ H NMR of 24 (400 MHz,CDCl₃) δ1.55 (s, 18H), 3.8 (s, 3H), 5.05 (d, 1H), 6.0 (d, 1H), 6.25 (d,1H), 6.3 (d, 1H), 6.9 (d, 2H), 7.1 (d, 4H), 7.32 (m, 4H), 7.45 (d, 1H),7.48 (d, 1H), 8.0 (d, 1H). ##STR47##

¹ H NMR of 25 (400 MHz, CD₃ OD) δ3.82 (s, 3H), 5.08 (d, 1H), 6.02 (d,1H), 6.4 (d, 2H), 6.9 (d, 2H), 7.22 (d, 4H), 7.42 (d, 4H), 7.6 (d, 2H),8.03 (d, 2H). MS ESI (neg ion) for M-H!⁻ : 487 (calculated 488).##STR48##

Hydroxyester 24 (1 equiv) was oxidized to ketoester 26 at 23° C. in CH₂Cl₂, in the presence of catalytic amount of TPAP (0.1 equiv),N-methylmorpholine oxide (2 equiv) and 4 Å activated powdered molecularsieves (500 mg/mol of substrate). ¹ H NMR of 26 (400 MHz, CDCl₃) δ1.55(s, 18H), 3.8 (s, 3H), 6.25 (d, 1H), 6.29 (d, 1H), 6.9 (d, 2H), 7.0 (s,1H), 7.5 (m, 10H), 7.95 (d, 1H), 8.02 (d, 1H). ##STR49##

¹ H NMR of 27 (400 MHz, CD₃ OD) δ3.82 (s, 3H), 6.45 (d, 1H), 6.55 (d,1H), 6.95 (d, 2H), 7.18 (s, 1H), 7.65 (m, 10H), 8.0 (d, 1H), 8.08 (d,1H).

Method 3 ##STR50##

By allowing an acid chloride (R₁ COCl) to react with an aldehyde (R₂CHO) wherein R₁, R₂ are defined as above in formula (A2) and bysubsequently oxidizing (A 10)-3.

The first step in this reaction may be carried out in a solvent or acombination of solvents such as tetrahydrofuran (THF), dichloromethane(CH₂ Cl₂), in the presence of a catalyst (e.g. TiCl₃), and a base (e.g.pyridine) at temperatures ranging from -78° C. to 23° C., for 1 to 60hours. The second step in this reaction may be carried out in a solventsuch as dichloromethane (CH₂ Cl₂), in the presence of an oxidizingreagent (e.g. tetrapropylammonium perruthenate (VII) (TPAP)) andactivated 4 Å molecular sieves at temperatures ranging from 0° C. to 23°C., for 1 to 60 hours.

EXAMPLES ##STR51##

Prepared according to Araneo et al (Tetrahedron Lett. 1994, 35, 2213).The reaction was stirred for 4 hrs at 23° C., the crude mixture wasseparated by flash chromatography (ethyl acetate in hexane eluent) toyield hydroxyester 28. ¹ H NMR of 28 (400 MHz, CDCl₃) δ1.55 (s, 18H),1.6 (m, 4H), 2.2-2.4 (m, 4H), 3.6 (s, 3H), 4.9 (d, 1H), 5.85 (d, 1H),6.25 (d, 1H), 6.3 (d, 1H), 7.07 (m, 4H), 7.3 (m, 4H), 7.45 (m, 2H).##STR52##

¹ H NMR of 29 (400 MHz, CD₃ OD) δ1.5 (m, 4H), 2.3 (m, 2H), 2.4 (m, 2H),3.6 (s, 3H), 4.95 (d, 1H), 5.85 (d, 1H), 6.4 (d, 2H), 7.2 (m, 4H), 7.42(d, 4H), 7.6 (d, 2H). MS ESI (neg ion) for M-H!⁻ : 495 (calculated 496).##STR53##

Hydroxyester 28 was oxidized to ketoester 30 as above. ¹ H NMR of 30(400 MHz, CDCl₃) δ1.55 (s, 18H), 1.65 (s br, 4H), 2.3 (m, 2H), 2.5 (m,2H), 3.6 (s, 3H), 6.3 (d, 1H), 6.35 (d, 1H), 6.78 (s, 1H), 7.4-7.6 (m,8H), 7.9 (d, 2H).

General method for the synthesis of compounds (A3)

Method 1 ##STR54##

By allowing a carboxylic acid (R₁ CO₂ H) to react with an isocyanide (R₂NC) and an aldehyde (R₃ CHO) wherein R₁, R₂, and R₃ are defined as abovein formula (A3).

These reactions may be carried out in a solvent or a combination ofsolvents such as dichloromethane (CH₂ Cl₂), chloroform (CHCl₃), methanol(MeOH), tetrahydrofuran (THF) or acetonitrile (CH₃ CN), in the presenceor absence of a catalyst (e.g. ZnCl₂, MgBr₂) at temperatures rangingfrom -78° C. to 80° C., for 1 to 60 hours.

EXAMPLES ##STR55##

Prepared according to Passerini (Gazz. Chim. Ital. 1926, 56, 826).

A solution of the carboxylic acid, aldehyde and isocyanide in a givensolvent selected from tetrahydrofuran, acetonitrile, ethyl ether orchloroform was stirred between 0° and 25° C. for 1 to 3 days. Thesolution was diluted with ethyl acetate, washed with saturated sodiumbicarbonate and dried over sodium sulfate. The solvent was removed invacuo and the residue was purified by silica gel chromatography.##STR56##

¹ H NMR of 32 (400 MHz, d₆ -acetone) δ0.8 (t, 3H), 1.1-1.6 (m, 9H), 1.97(m, 1H), 3.9 (m, 2H), 4.1 (m, 2H), 5.3 (t, 1H), 6.62 (d, 1H), 7.7 (d,1H), 7.8 (d, 2H), 8.05 (d, 2H).

Method 2 ##STR57##

By allowing a carboxylic acid (R₁ CO₂ H) and an aldehyde (R₃ CHO) toreact with a polymer bound isocyanide (R₂ NC) wherein R₁, R₂, and R₃ aredefined as above in formula (A3).

These reactions may be carried out on functionalized cross linkedpolystyrene polymers such as Merrifield resin, Wang resin, Rink resin,"TENTAGEL™" which is a polyethylene-polystyrene polymer resin, in asolvent or a combination of solvents such as dichloromethane (CH₂ Cl₂),chloroform (CHCl₃), methanol (MeOH), tetrahydrofuran (THF), acetonitrile(CH₃ CN), in the presence or absence of a catalyst (e.g. ZnCl₂, MgBr₂)at temperatures ranging from -78° C. to 80° C., for 1 to 60 hours. Theproduct maybe released from the polymer by conditions known to thoseskilled in the art.

EXAMPLES ##STR58##

Prepared according to Zhang et al (Tetrahedron Letters 1996, 37, 751).##STR59##

A solution of the carboxylic acid 3 in tetrahydrofuran was added to amixture of the aldehyde and isocyanide resin 33 in tetrahydrofuran oracetonitrile. The mixture was stirred at 25° C. or 60° C. for 1 to 3days. The resin was filtered and washed with dichloromethane andmethanol and dried. Compounds 34 were isolated after treatment of theresin with a solution of 50% trifluoroacetic acid in dichloromethane for1 hour at 23° C. and removal of the solvent in vacuo. ##STR60##

A solution of the carboxylic acid in tetrahydrofuran was added to amixture of the aldehyde 4 isocyanide resin 33 in tetrahydrofuran oracetonitrile. The mixture was stirred at 25° C. or 60° C. for 1 to 3days. The resin was filtered and washed with dichloromethane andmethanol and dried. Compounds 35 were isolated after treatment of theresin with a solution of 50% trifluoroacetic acid in dichloromethane for1 hour at 23° C. and removal of the solvent in vacuo.

                  TABLE 1                                                         ______________________________________                                         ##STR61##                                                                                                MWt      MH!.sup.-                                Compound R.sub.2    n       (Calculated)                                                                          (Found)                                   ______________________________________                                        36                                                                                      ##STR62## 2       475;477 474;476                                   37                                                                                      ##STR63## 5       519     518                                       38       C.sub.6 H.sub.14 CHO                                                                     2       405     404                                       39       C.sub.6 H.sub.14 CHO                                                                     5       447     446                                       40       C.sub.9 H.sub.20 CHO                                                                     2       447     446                                       41       C.sub.9 H.sub.20 CHO                                                                     5       489     488                                       ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                         ##STR64##                                                                    Com-                          MWt      MH!.sup.-                              pound  R.sub.1          n     (Calculated)                                                                          (Found)                                 ______________________________________                                        42                                                                                    ##STR65##       2     427     426                                     43                                                                                    ##STR66##       5     469     468                                     44                                                                                    ##STR67##       2     442     441                                     45                                                                                    ##STR68##       2     441     440                                     46                                                                                    ##STR69##       5     483     482                                     47                                                                                    ##STR70##       2     419     418                                     48                                                                                    ##STR71##       5     519     518                                     49                                                                                    ##STR72##       2     417     416                                     50                                                                                    ##STR73##       5     459     458                                     ______________________________________                                    

General method for the synthesis of compounds (A4) ##STR74##

By allowing an acid chloride (R₁ COCl) to react with an aldehyde (R₂CHO) wherein R₁, R₂ are defined as above in formula (A4).

These reactions may be carried out in a solvent or combination ofsolvents such as tetrahydrofuran (THF), dichloromethane (CH₂ Cl₂), inthe presence of a catalyst (e.g. TiCl₃), and a base (e.g. pyridine) attemperatures ranging from -78° C. to 23° C., for 1 to 60 hours.

Example ##STR75##

Prepared according to Araneo et al (Tetrahedron Lett. 1994, 35, 2213). ¹H NMR of 51 (400 MHz, CDCl₃) δ1.45 (s, 9H), 1.5 (m, 4H), 2.1-2.3 (m,4H), 3.6) s, 3H), 4.6 (s, 1H), 6.25 (d, 1H), 6.97 (d, 2H), 7.25 (d, 2H),7.5 (d, 1H).

General methods for the synthesis of compounds (A6)

Method 1 ##STR76##

By allowing a compound of formula (A5) to react with an aldehyde (R₂CHO), a primary amine (R₁ NH₂) and ammonium acetate wherein R₁, R₂, R₃and R₄ are defined as above in formula (A6).

These reactions may be carried out in a solvent such as acetic acid(AcOH) at temperatures ranging from 23° C. to 120° C., for 1 to 60hours.

EXAMPLES ##STR77##

Prepared according to Krieg et al (Z Naturforsch teil 1967, 22b, 132).

To 47 mg of 6 (0.1 mmol, 1.0 equiv), R₂ CHO (0.1 mmol, 1.0 equiv) in 1mL of acetic acid was added 231 mg of ammonium acetate (3.0 mmol, 30equiv) in 0.5 mL of acetic acid and the mixture was placed in 100° C.preheated oil bath for 1 hour. The solution was then poured into etherand washed with saturated sodium bicarbonate. The organic layer wasdried over sodium sulfate, filtered and concentrated in vacuo to yieldthe desired imidazoles 52 which were purified by preparative thin layerchromatography with ethyl acetate-hexane or methanol-dichloromethane aseluent.

                  TABLE 3                                                         ______________________________________                                         ##STR78##                                                                     ##STR79##                                                                                            MWt.    MWt.                                          Entry                                                                              R.sub.2            (Calc.) (Obs.)                                        ______________________________________                                        54                                                                                  ##STR80##         480     479                                           55   H                  --*     --*                                           56                                                                                  ##STR81##         525     526                                           57                                                                                  ##STR82##         481     482                                           58                                                                                  ##STR83##         --*     --*                                           59                                                                                  ##STR84##         511     512                                           60                                                                                  ##STR85##         --*     --*                                           61                                                                                  ##STR86##         --*     --*                                           62                                                                                  ##STR87##         521     522                                           63                                                                                  ##STR88##         487     488                                           64                                                                                  ##STR89##         533     534                                           65                                                                                  ##STR90##         --*     --*                                           66                                                                                  ##STR91##         --*     --*                                           67                                                                                  ##STR92##         --*     --*                                           68                                                                                  ##STR93##         --*     --*                                           69                                                                                  ##STR94##         482     483                                           70                                                                                  ##STR95##         442     441                                           71                                                                                  ##STR96##         494     495                                           72                                                                                  ##STR97##         526     527                                           73                                                                                  ##STR98##         --*     --*                                           74                                                                                  ##STR99##         489     490                                           75   Ph                 436     435                                           76                                                                                  ##STR100##        --*     --*                                           77   n-C.sub.5 H.sub.11 --*     --*                                           78                                                                                  ##STR101##        454     455                                           79                                                                                  ##STR102##        --*     --*                                           80                                                                                  ##STR103##        560     561                                           81                                                                                  ##STR104##        536     537                                           82                                                                                  ##STR105##        --*     --*                                           83                                                                                  ##STR106##        --*     --*                                           84                                                                                  ##STR107##        526     527                                           85                                                                                  ##STR108##        480     481                                           86                                                                                  ##STR109##        494     495                                           87                                                                                  ##STR110##        542     543                                           88                                                                                  ##STR111##        561     562                                           89                                                                                  ##STR112##        476     477                                           90                                                                                  ##STR113##        493     494                                           ______________________________________                                         * "--": data not available.                                              

54 (400 MHz, CDCl₃ --CD₃ OD 10:1) δ6.23 (d, 2H), 7.3-7.48 (m, 10H), 7.88(d, 2H), 8.02 (d, 2H).

55 (400 MHz, CD₃ OD) δ6.5 (d, 2H), 7.52 (d, 4H), 7.7 (m, 6H), 9.1(s,1H).

56 (400 MHz, CD₃ OD) δ6.3 (s, 2H), 6.52 (d, 2H), 7.4-7.9 (m, 12H).

57 (400 MHz, CD₃ OD) δ6.52 (d, 2H), 7.50-8.36 (m, 14H).

58 (400 MHz, CDCl₃ --CD₃ OD 10:1) δ3.7 (s, 3H), 6.3 (d, 2H), 6.85 (d,2H), 7.4 (m, 8H), 7.5 (d, 2H), 7.85 (d, 2H).

59 (400 MHz, CD₃ OD) δ3.98 (s, 3H), 6.52 (d, 2H), 7.50-7.76 (m, 13H).

60 (400 MHz, CDCl₃ --CD₃ OD 10:1) δ6.3 (d, 2H), 6.5 (br s, 1H), 6.85 (d,2H), 7.3-7.6 (m, 12H).

61 di-tert-butyl ester (400 MHz, CDCl₃ --CD₃ OD 6:1) δ1.4 (s, 18H), 6.2(d, 2H), 6.9 (t, 1H), 7.2-7.42 (m, 11H), 7.58 (d, 1H), 7.62 (d, 1H).

62 (400 MHz, CD₃ OD) δ6.50 (d, 2H), 7.30-8.70 (m, 12H).

63 (400 MHz, CD₃ OD) δ6.54 (d, 2H), 7.46-8.60 (m, 16H).

64 (400 MHz, CD₃ OD) δ3.90 (s, 3H), 6.50 (d, 2H), 7.50-8.30 (m, 13H).

65 di-tert-butyl ester (400 MHz, CDCl₃ --CD₃ OD 6:1) δ1.4 (s, 18H), 6.2(d, 2H), 6.65 (t, 1H), 7.3-7.5 (m, 12H).

66 di-tert-butyl ester (400 MHz, CDCl₃ --CD₃ OD 6:1) δ1.4 (s, 18H), 6.2(dd, 2H), 7.1 (q, 1H), 7.3-7.5 (m, 10H), 7.6 (br d, 1H), 7.7 (dd, 1H).

67 di-tert-butyl ester (400 MHz, CDCl₃ --CD₃ OD 6:1) δ1.4 (s, 18H), 6.2(d, 2H), 7.1 (t, 2H), 7.3-7.5 (m, 10H), 7.75 (m, 1H).

68 (400 MHz, CD₃ OD) δ1.2 (t, 6H), 3.48 (q, 4H), 6.52 (d, 2H), 7.3-8.02(m, 15H).

69 (400 MHz, CD₃ OD) δ3.96 (s, 3H), 6.52 (d, 2H), 7.04-7.70 (m, 13H).

70 (400 MHz, CD₃ OD) δ6.46 (d, 2H), 7.14 (d, 1H), 7.50-7.80 (m, 12H).

71 (400 MHz, CD₃ OD) δ4.32 (m, 4H), 6.52 (d, 2H), 7.1-7.7 (m, 13H).

72 (400 MHz, CD₃ OD) δ3.90-4.02 (3s, 9H), 6.50 (d, 2H), 6.90-7.80 (m,12H).

73 (400 MHz, CD₃ OD) δ6.5 (d, 2H), 7.55 (d, 4H), 7.65 (m, 6H), 7.95 (d,2H). 8.15 (d, 2H).

74 (400 MHz, CD₃ OD) δ3.95 (s, 3H), 6.56 (d, 2H), 6.9-7.82 (m, 14H).

75 (400 MHz, CDCl₃ --CD₃ OD 10:1) δ6.34 (d, 2H), 7.3-7.4 (m, 11H), 7.52(d, 2H), 8.92 (d,2H).

76 di-tert-butyl ester (400 MHz, CDCl₃ --CD₃ OD 6:1) δ1.4 (s, 18H), 6.2(br d, 2H), 6.9 (m, 1H), 7.05 (m, 1H), 7.3-7.5 (m, 10H).

77 (400 MHz, CDCl₃ --CD₃ OD 6:1) δ0.9 (m, 5H), 1.3 (m, 2H), 1.7 (m, 2H),2.9 (t, 2H), 6.35 (d, 2H), 7.3-7.6 (m, 10H).

78 (400 MHz, CD₃ OD) δ6.50 (d, 2H), 7.40-7.9 (m, 14H).

79 di-tert-butyl ester (400 MHz, CDCl₃) δ1.4 (s, 18H), 6.3 (d, 2H), 7.1(d, 2H), 7.22 (d, 1H), 7.34 (t, 2H), 7.4-7.7 (m, 14H), 7.9 (d, 2H).

80 (400 MHz, CD₃ OD) δ6.54 (d, 2H), 7.6-8.0 (m, 19H).

81 (400 MHz, CD₃ OD) δ6.54 (d, 2H), 7.6-8.90 (m, 19H).

82 (400 MHz, CD₃ OD) δ6.50 (d, 2H), 7.58-8.0 (m, 14H).

83 (400 MHz, CD₃ OD) δ3.96 (s, 3H), 6.52 (d, 2H), 7.36-7.90 (m, 13H).

84 (400 MHz, CD₃ OD) δ6.50 (d, 2H), 7.55-7.70 (m, 10H).

85 (400 MHz, CD₃ OD) δ6.12 (s, 2H), 6.56 (d, 2H), 7.10-7.60 (m, 13H).

86 (400 MHz, CD₃ OD) δ2.20 (s, 3H), 2.40 (s, 3H), 3.90 (s, 3H), 6.52 (d,2H), 7.10-7.70 (m, 12H).

87 (400 MHz, CD₃ OD) δ5.20 (s, 2H), 6.56 (d, 2H), 7.22-7.98 (m, 19H).

88 (400 MHz, CD₃ OD) δ1.52 (2s, 12H), 1.74 (s, 4H), 2.42 (s, 3H), 6.52(d, 2H), 7.40-7.68 (m, 13H).

89 (400 MHz, CD₃ OD) δ1.12 (t, 2H), 3.0 (m, 4H), 6.56 (d, 2H), 7.52-7.62(m, 13H).

90 (400 MHz, CD₃ OD) δ2.14 (s, 3H), 6.54 (d, 2H), 7.58-8.0 (m, 14H).##STR114##

Prepared according to Krieg et al (Z Naturforsch teil 1967, 22b, 132).

¹ H NMR of 91 (400 MHz, CD₃ OD) δ3.9 (s,3H), 6.2 (d, 2H), 6.95 (d, 2H),7.2 (d, 2H), 7.4-7.6 (m, 6H), 7.9 (d, 2H). ##STR115##

Prepared according to Krieg et al (Z Naturforsch teil 1967, 22b, 132).

                                      TABLE 4                                     __________________________________________________________________________     ##STR116##                                                                                                     MWt    MWt                                  Entry                                                                              R.sub.1         R.sub.2      (Calc.)                                                                              (Obs.)                               __________________________________________________________________________     94  n-C.sub.4 H.sub.9                                                                             H            416    415                                   95  n-C.sub.4 H.sub.9                                                                              ##STR117##  498    497                                   96  Ph                                                                                             ##STR118##  518    517                                   97  n-C.sub.4 H.sub.9                                                                              ##STR119##  522    521                                   98  Ph                                                                                             ##STR120##  542    541                                   99  Ph              H            436    435                                  100                                                                                 ##STR121##                                                                                    ##STR122##  582    581                                  __________________________________________________________________________

94 (400 MHz, CD₃ OD) δ0.8 (t, 3H), 1.22 (m, 2H), 1.62 (m, 2H), 4.10 (t,2H), 6.42 (d, 1H), 6.58 (d, 1H), 7.32-7.80 (m, 10H), 9.18 (s, 1H).

95 (400 MHz, CD₃ OD) δ0.64 (t, 3H), 1.04 (m, 2H), 1.58 (m, 2H), 4.20 (t,2H), 6.42 (d, 1H), 6.62 (d, 1H), 7.42-8.0 (m, 13H).

96 (400 MHz, CD₃ OD) δ6.42 (2d, 2H), 7.12-7.68 (m, 18H).

97 (400 MHz, CD₃ OD) δ0.6 (t, 3H), 1.0 (m, 2H), 1.38 (m, 2H), 4.12 (t,2H), 3.84 (s, 3H), 6.42 (d, 1H), 6.62 (d, 1H), 7.22-7.8 (m, 13H).

98 (400 MHz, CD₃ OD) δ3.80 (s, 3H), 6.44 (2d, 2H), 6.94-7.68 (m, 19H).

99 (400 MHz, CD₃ OD) δ6.44 (2d, 2H), 7.20-7.60 (m, 15H), 9.2 (s, 1H).

100 (400 MHz, CD₃ OD) δ1.22 (s, 9H), 2.40 (s, 3H), 6.36-6.44 (2d, 2H),7.26-7.60 (m, 18H).

Method 2 ##STR123##

By allowing a polymer bound compound of formula (A5)-2 to react with analdehyde (R₂ CHO), a primary amine (R₁ NH₂) and ammonium acetate whereinR₁, R₂, R₃ and R₄ are defined as above in formula (A6).

These reactions may be carried out on functionalized cross linkedpolystyrene polymers such as Merrifield resin, Wang resin, Rink resin,"TENTAGEL™" which is a polyethylene-polystyrene polymer resin, in asolvent such as acetic acid (AcOH) at temperatures ranging from 23° C.to 120° C., for 1 to 60 hours. The product maybe released from thepolymer using conditions known to those skilled in the art.

EXAMPLES ##STR124##

To resin 17 were added excess NH₄ OAc and R₂ CHO and acetic acid and themixture was heated at 100° C. for 15 hours, cooled to 23° C. and washedwith methanol and dichloromethane and dried under vacuum. Thetrifluoroacetate salts of imidazoles 101 were isolated followingtreatment of the resin with a solution of 20% trifluoroacetic acid indichloromethane for 20 minutes at 23° C. ##STR125##

                                      TABLE 5                                     __________________________________________________________________________                                MWt.                                                                              MWt.                                          Entry                                                                             R.sub.1     R.sub.2     (Calc.)                                                                           (Obs.)                                        __________________________________________________________________________    103 Me                                                                                                    495 496                                           104                                                                                ##STR126##                                                                                ##STR127## 620 621                                           105                                                                                ##STR128##                                                                                ##STR129## 602 603                                           106                                                                                ##STR130##                                                                                ##STR131## 586 587                                           107                                                                                ##STR132##                                                                                ##STR133## 638 639                                           108                                                                                ##STR134##                                                                                ##STR135## 634 635                                           109 2-propyl                                                                                   ##STR136## 496 497                                           110 2-propyl                                                                                   ##STR137## 523 524                                           111 2-propyl                                                                                   ##STR138## 553 554                                           112 2-indanyl                                                                                  ##STR139## 627 628                                           113                                                                                ##STR140##                                                                                ##STR141## 626 627                                           __________________________________________________________________________     ##STR142##                                                                

Same procedure as imidazoles 101. ##STR143##

Same procedure as imidazoles 101.

                                      TABLE 6                                     __________________________________________________________________________     ##STR144##                                                                                                         MWt.     MWt.                           Entry  R.sub.1         R.sub.2        (Calc.)  (Obs.)                         __________________________________________________________________________    116                                                                                   ##STR145##                                                                                    ##STR146##    559      560                            117                                                                                   ##STR147##                                                                                    ##STR148##    562      563                            118                                                                                   ##STR149##                                                                                    ##STR150##    633      634                            119                                                                                   ##STR151##                                                                                    ##STR152##    642      643                            120                                                                                   ##STR153##                                                                                    ##STR154##    592      593                            121                                                                                   ##STR155##                                                                                    ##STR156##    579      580                            122    n-propyl                                                                                       ##STR157##    508      509                            123    n-propyl                                                                                       ##STR158##    562, 564 563, 565                       124    n-butyl                                                                                        ##STR159##    528      529                            125    n-heptyl                                                                                       ##STR160##    579      580                            126    n-octyl                                                                                        ##STR161##    566      567                            127    n-octyl                                                                                        ##STR162##    619      620                            128                                                                                   ##STR163##                                                                                    ##STR164##    612      613                            __________________________________________________________________________

Method 3 ##STR165##

By allowing a compound of formula (129) (J. Org. Chem., 1995, 60, 8231;J. Org. Chem., 1993, 58, 4785) to react with an aldehyde (R₂ CHO), aprimary amine (R₁ NH₂) and ammonium acetate wherein R₁, R₂, R₃ and R₄are defined as above in formula (A6).

These reactions may be carried out in a solvent such as acetic acid(AcOH) at temperatures ranging from 23° C. to 120° C., for 1 to 60hours.

EXAMPLES ##STR166##

Prepared according to Wasserman et al (J. Org. Chem., 1995, 60, 8231; J.Org. Chem., 1993, 58, 4785). Benzyl (triphenylphosphoranylidene) acetate(130) was purchased from Aldrich chemical company and used directly. ¹ HNMR of 131 (400 MHz, CDCl₃) δ1.5 (s, 9H), 4.62 (s, 2H), 6.3 (d, 1H),6.62 (d, 2H), 7.05 (t, 2H), 7.1 (t, 1H), 7.38-7.8 (m, 20H). TLC: R_(f)=0.5 (30% ethyl acetate-hexane). ##STR167##

Prepared according to Wasserman et al (J. Org. Chem., 1995, 60, 8231; J.Org. Chem., 1993, 58, 4785). ¹ H NMR of 132 (400 MHz, CDCl₃) δ1.5 (s,9H), 5.1 (s, 2H), 5.15 (br s, 2H, 2×H--O), 6.4 (d, 1H), 6.95 (d, 2H),7.1 (t, 2H), 7.18 (t, 1H), 7.4 (d, 2H), 7.5 (d, 1H), 7.9 (d, 2H). TLC:R_(f) =0.7 (30% ethyl acetate-hexane). ##STR168##

¹ H NMR of 133 (400 MHz, CDCl₃ --CD₃ OD, 8:1) δ5 (s, 2H), 6.4 (d 1H),6.9-7.16 (m, 5H), 7.35 (d, 2H), 7.53 (d, 1H), 7.9 (d, 2H). ##STR169##

Prepared according to Brackeen et al (Tetrahedron Letters 1994, 35,1635). For other approaches to imidazole-4-carboxylates see: a) Nunamiet al (J. Org. Chem. 1994, 59, 7635). b) Heindel et al (TetrahedronLetters 1971, 1439). ¹ H NMR of 134 (400 MHz, 8:1 CDCl₃ --CD₃ OD) δ5.2(s, 2H), 6.4 (d, 1H), 7.25 (br s, 5H), 7.5 (d, 2H), 7.6 (d, 1H), 7.7 (d,2H), 8.3 (s, 1H).

Method 4 ##STR170##

By allowing a compound of formula (129) to react with a polymer boundaldehyde (R₁ CHO), a primary amine (R₂ NH₂) and ammonium acetate whereinR₁, R₂, R₃ and R₄ are defined as above in formula (A6).

This reaction may be carried out on functionalized cross linkedpolystyrene polymers such as Merrifield resin, Wang resin, Rink resin,Tentagel™ resin, in a solvent such as acetic acid (AcOH) at temperaturesranging from 23° C. to 120° C., for 1 to 60 hours. The product maybereleased from the polymer using conditions known to those skilled in theart.

EXAMPLES ##STR171##

For leading references see: a) Mathias (Synthesis, 1979, 561). b)Sarantakis et al (Biochem. Biophys. Res. Commun. 1976, 73, 336). c)Hudson et al (Peptide Chemistry 1985 (Kiso, Y., ed.), 1986, ProteinResearch Foundation, Osaka.). d) Wang (J. Am. Chem. Soc. 1973, 95,1328). e) Lu et al (J. Org. Chem. 1981, 46, 3433). To 6 mmol (1 equiv)of Wang resin in 130 mL of dry dimethylformamide was added 18 mmol (3equiv) of diisopropylcarbodiimide and the mixture was sonnicated for 4hours (final bath temperature was 37° C). 4-Formylcinnamic acid (18mmol, 3 equiv) and 4-dimethylaminopyridine (6 mmol, 1 equiv) were addedand the mixture was magnetically stirred for 48 hours at ambienttemperature. The resin was filtered and thoroughly washed withdimethylforamide (500 mL), methanol (500 mL), dichloromethane (500 mL)and methanol (500 mL) and dried in vacuo (0.1 mmHg) for 24 hours. Acoupling yield of 80% was established by cleaving 100 mg of the resinwith a solution of 20% trifluoroacetic acid in dichloromethane for 20min at ambient temperature. ##STR172##

To 60 mg (0.048 mmol, 1.0 equiv) of 135 was added 40 mg (0.097 mmol, 2.0equiv) of 132 followed by 37 mg (0.481 mmol, 5.0 equiv) of ammoniumacetate and 0.2 mL of acetic acid. The mixture was heated to 100° C. for15 hours, filtered, washed with dimethylformamide, dichloromethane,methanol and dichloromethane. The crude product was isolated bytreatment of the polymer with a solution of 50% trifluoroacetic acid indichloromethane for 1 hour at 23° C. The solvent was removed and theresidue was purified by preparative thin layer chromatography (20%methanol-dichloromethane eluent). ¹ H NMR of 136 (400 MHz, CD₃ OD) δ5.15(s, 2H), 6.48 (d, 1H), 6.55 (d, 1H), 7.25 (br s, 4H), 7.5-7.8 (m, 9H),8.1 (d, 1H). MS (ESI negative ion) M-H!⁻ : 493;

Method 5 ##STR173##

By allowing a primary amine (R₁ NH₂), a carboxylic acid (R₂ CO₂ H) and aketoaldehyde (R₄ COCHO) to react with a polymer bound isocyanide (R₃ NC)and by subsequently cyclizing compound 137 with ammonium acetate whereinR₁, R₂, R₃ and R₄ are defined as above in formula (A6).

The first step in this reaction may be carried out on functionalizedcross linked polystyrene resins such as Merrifield resin, Wang resin,Rink resin, "TENTAGEL™" which is a polyethylene-polystyrene polymerresin, in a solvent or a combination of solvents such as dichloromethane(CH₂ Cl₂), chloroform (CHCl₃), methanol (MeOH), tetrahydrofuran (THF) oracetonitrile (CH₃ CN), in the presence or absence of a catalyst (e.g.ZnCl₂, MgBr₂) at temperatures ranging from -78° C. to 80° C., for 1 to60 hours. The second step in this reaction may be carried out in asolvent such as acetic acid (AcOH) at temperatures ranging from 23° C.to 120° C., for 1 to 60 hours. ##STR174##

Prepared according to Gunn et al (J. Org. Chem. 1977, 42, 754).##STR175##

Prepared according to Zhang et al (Tetrahedron Letters 1996, 37, 751).##STR176##

Prepared according to Zhang et al (Tetrahedron Letters 1996, 37, 751).

¹ H NMR of mono tert-butyl ester of 140 (400 MHz, CDCl₃) δ1.1 (m,2H),1.2 (d,3H), 1.3 (m,2H), 1.5 (s,9H), 1.56 (m,2H), 2.2 (m,2H), 2.9 (m,1H),3.1 (m,1H), 3.2 (m,1H), 3.8 (s,3H), 4.6 (m,2H), 6.1 (d,1H), 6.9 (t,4H),7.1 (m,5H), 7.4 (d, 2H), 7.6 (d,1H).

Method 6 ##STR177##

By allowing a carboxylic acid (R₁ CO₂ H) to react with an isocyanide (R₃NC) and a ketoaldehyde (R₂ COCHO) and by allowing compound 141 tocyclize in the presence of ammonium acetate, wherein R₁, R₂, and R₃ aredefined as above in formula (A6).

The first step in this reaction may be carried out in a solvent or acombination of solvents such as dichloromethane (CH₂ Cl₂), chloroform(CHCl₃), methanol (MeOH), tetrahydrofuran (THF), acetonitrile (CH₃ CN),in the presence or absence of a catalyst (e.g. ZnCl₂, MgBr₂) attemperatures ranging from -78° C. to 80° C., for 1 to 60 hours. Thesecond step in this reaction may be carried out in a solvent such asacetic acid (AcOH) at temperatures ranging from 23° C. to 120° C., for 1to 60 hours.

EXAMPLES ##STR178##

Prepared according to Bossio et al (Liebigs Ann. Chem. 1991, 1107).

To an ethyl ether mixture of the carboxylic acid and ketoaldehyde at 0°C. was added dropwise an ethyl ether solution of the isocyanide. Themixture was warmed to 25° C. and stirred for 2 hours to 3 days. Thesolution was diluted with ethyl acetate, washed with saturated sodiumbicarbonate and dried over sodium sulfate. The solvent was removed invacuo and the residue was purified by silica gel chromatography to yield(α-Acyloxy-β-ketoamide 142.

A solution of the (α-Acyloxy-β-ketoamide 142 (1 equiv) and ammoniumacetate (30 equiv) in acetic acid was heated at 100° C. for 2 to 15hours. The reaction was cooled to 23° C., diluted with ethyl acetate,washed with saturated sodium bicarbonate and dried over sodium sulfate.Solvent was removed in vacuo and the crude mixture was separated bysilica gel chromatography to provide imidazole 143. ##STR179##

¹ H NMR of 144 (400 MHz, d₆ -acetone) δ0.85 (t, 3H), 1.2-1.6 (m, 4H),3.3 (m, 2H), 6.55 (s, 1H), 6.62 (d, 1H), 7.3 (t, 2H), 7.7 (d, 1H), 7.82(d, 2H), 8.1 (d, 2H), 8.25 (dd, 2H). ##STR180##

¹ H NMR of 146 (400 MHz, d₆ -acetone) δ0.9 (t, 3H), 1.4 (m, 2H), 1.6 (m,2H), 3.35 (m, 2H), 6.58 (d, 1H), 7.12 (t, 2H), 7.65 (d, 1H), 7.78 (d,2H), 8.1 (s br, 1H), 8.05 (m, 1H), 8.2 (d, 2H).

General method for the synthesis of compounds (A7) ##STR181##

By allowing a carboxylic acid (R₁ CO₂ H) to react with an isocyanide (R₃NC) and a ketoaldehyde (R₂ COCHO) wherein R₁, R₂, and R₃ are defined asabove in formula (A7) and by allowing compound 141 to cyclize in thepresence of ammonium acetate.

The first step in this reaction may be carried out in a solvent or acombination of solvents such as dichloromethane (CH₂ Cl₂), chloroform(CHCl₃), methanol (MeOH), tetrahydrofuran (THF), acetonitrile (CH₃ CN),in the presence or absence of a catalyst (e.g. ZnCl₂, MgBr₂) attemperatures ranging from -78° C. to 80° C., for 1 to 60 hours. Thesecond step in this reaction may be carried out in a solvent such asacetic acid (AcOH) at temperatures ranging from 23° C. to 120° C., for 1to 60 hours.

EXAMPLES ##STR182##

Prepared according to Bossio et al (Liebigs Ann. Chem. 1991, 1107).

A solution of the (α-Acyloxy-β-ketoamide 141 (1 equiv) and ammoniumacetate (2 equiv) in acetic acid was heated at 100° C. for 2 to 15hours. The reaction was cooled to 23° C., diluted with ethyl acetate,washed with saturated sodium bicarbonate and dried over sodium sulfate.Solvent was removed in vacuo and the crude oxazole 147 was purified bysilica gel chromatography. ##STR183##

¹ H NMR of 148 (400 MHz, d₆ -acetone) δ0.9 (t, 3H), 1.4 (m, 2H), 1.6 (m,2H), 3.42 (m, 2H), 6.63 (d, 1H), 7.2 (t, 2H), 7.7 (d, 1H), 7.9 (d, 2H),8.18 (s br, 1H), 8.25 (d, 2H), 8.6 (m, 1H).

General methods for the synthesis of compounds (A8) and (A9)

Method 1 ##STR184##

By allowing a compound of formula (A5) to react with compound of formula(149) wherein R₁, R₂, R₃, R₄, R₅, and R₆ are defined as above in formula(A8).

These reactions may be carried out in a solvent or a combination ofsolvents such as dioxane or acetic acid (AcOH) at temperatures rangingfrom 23° C. to 120° C., for 1 to 60 hours.

EXAMPLES ##STR185##

A solution of 0.1 mmol of diamine 150 and 0.1 mmol of 6 in 1.2 mL of1,4-dioxane-acetic (5:1) was heated at 100° C. Upon completion of thereaction as judged by thin layer chromatography, ethyl acetate was addedand the organic layer was washed with water, 0.5M citric acid, 10%sodium bicarbonate and dried over sodium sulfate compounds were purifiedusing silica gel chromatography.

                  TABLE 7                                                         ______________________________________                                         ##STR186##                                                                    ##STR187##                                                                    ##STR188##                                                                   Compound     R.sub.1                                                                             R.sub.2    R.sub.3                                                                             R.sub.4                                   ______________________________________                                        152          H     H          NO.sub.2                                                                            H                                         153          H     Cl         Cl    H                                         154          H     H          CH.sub.3                                                                            H                                         155          H     H          CO.sub.2 H                                                                          H                                         156          H     H          CO.sub.2 Me                                                                         H                                         157          H     H          H     H                                         ______________________________________                                    

152 (400 MHz, CD₃ OD) δ6.5 (d, 2H), 7.3 (s, 1H), 7.4-7.8 (m, 10H), 7.9(s, 1H), 8.05 (d, 1H). MS ESI (pos ion) for M+H!⁺ : 468 (calculated467).

153 (400 MHz, CD₃ OD) δ6.48 (d, 2H), 7.5 (dd, 8H), 7.6 (d, 2H), 8.24 (s,2H). MS ESI (pos ion) for M+H!⁺ : 491, 492 (calculated 490, 491).

154 (400 MHz, CD₃ OD) δ3.3 (s, 3H), 6.5 (d, 2H), 7.59 (s, 8H), 7.62 (d,2H), 8.3 (d, 1H), 8.55 (d, 1H), 8.95 (s, 1H). MS ESI (pos ion) for M+H!⁺: 437 (calculated 436).

155 (400 MHz, d₆ -DMSO) δ6.56 (d, 2H), 7.5 (m, 6H), 7.65 (d, 4H), 8.2(d, 1H), 8.3 (d, 1H), 8.6 (s, 1H). MS ESI (neg ion) for M-H!⁻ : 465(calculated 466).

156 (400 MHz, CD₃ OD) δ6.56 (d, 2H), 7.5 (s br, 8H), 7.65 (d, 2H), 8.2(d, 1H), 8.3 (d, 1H), 8.7 (s, 1H). MS ESI (neg ion) for M-H!⁻ : 479(calculated 480).

157 (400 MHz, d₆ -DMSO) δ6.52 (d, 2H), 7.54-8.16 (m, 14H).

Method 2 ##STR189##

By allowing a compound of formula (A5) to react with compound of formula(158) wherein R₁, R₂, R₃, R₄, R₅ are defined as above in formula (A9).

These reactions may be carried out in a solvent or a combination ofsolvents such as dioxane or acetic acid (AcOH) at temperatures rangingfrom 23° C. to 120° C., for 1 to 60 hours.

EXAMPLES ##STR190##

¹ H NMR of 160 (400 MHz, CD₃ OD) δ6.5 (d, 2H), 7.5-7.7 (m, 12H), 7.95(m, 1H), 8.65 (d, 1H), 9.15 (s, 1H).

MS ESI (pos ion) for M+H!⁺ : 424 (calculated 423).

General method for the synthesis of compounds (A10) ##STR191##

By allowing a compound of formula (A10)-1 prepared as above to reactwith a carboxylic acid (R₂ CO₂ H) wherein R₁ and R₂ are defined as abovein formula (A 10).

These reactions may be carried out in a solvent such as tetrahydrofuran(THF), dichloromethane (CH₂ Cl₂), in the presence of diisopropylcarbodiimide (DIC) and a base (e.g. 4,4-dimethylaminopyridine) attemperatures ranging from 0° C. to 23° C., for 1 to 60 hours.

EXAMPLES ##STR192##

To 50 mg of diol 22 in 1 mL of dichloromethane was added diisopropylcarbodiimide (2.2 equiv) and the reaction was stirred for 1 hour at 23°C. To the solution was added 4,4-dimethylaminopyridine (0.2 equiv)followed by para-methoxybenzoic acid (2.2 equiv) in 5 mL oftetrahydrofuran and the mixture was stirred for an additional 3 hours at23° C. The reaction was diluted with ethyl acetate and washed with 1NHCl, saturated sodium bicarbonate and the organic layer was dried oversodium sulfate. The crude mixture was purified using radialchromatography (ethyl acetate-hexane eluent). ¹ H NMR of 161 (400 MHz,CDCl₃) δ1.5 (s, 18H), 3.8 (s, 6H), 6.25 (d, 2H), 6.32 (s, 2H), 6.85 (d,4H), 7.18 (d, 4H), 7.31 (d, 4H), 7.45 (d, 2H), 7.95 (d, 4H). ##STR193##

¹ H NMR of 162 (400 MHz, CD₃ OD) δ3.8 (s, 6H), 6.4 (m, 4H), 6.95 (d,4H), 7.38 (d, 4H), 7.5 (d, 4H), 7.6 (d, 2H), 7.95 (d, 4H). MS ESI (negion) for M-H!⁻ : 621 (calculated 622).

General method for the synthesis of compounds (A11) ##STR194##

By allowing a compound of formula (A 10)-1 prepared as above to reactwith a sulfonyl chloride (R₂ SO₂ Cl), and subsequently by oxidizingintermediate 163 and by allowing intermediate 164 to react with athioamide (R₃ C(S)NH₂) wherein R₁, R₂ and R₃ are defined as above informula (A11).

The first step in this sequence of reactions may be carried out in asolvent such as tetrahydrofuran (THF), dichloromethane (CH₂ Cl₂), in thepresence of a base (e.g. 4,4-dimethylaminopyridine, triethylamine,triisopropylamine) and a sulfonyl chloride (e.g. tosyl chloride, mesylchloride), at temperatures ranging from -20° C. to 23° C., for 1 to 60hours. The second step in this sequence of reactions may be carried outin a solvent such as dichloromethane (CH₂ Cl₂), in the presence of anoxidizing reagent (e.g. tetrapropylammonium perruthenate (VII) (TPAP))and activated 4 Å molecular sieves at temperatures ranging from 0° C. to23° C., for 1 to 60 hours. The third step in this sequence of reactionsmay be carried out in a solvent such as acetic acid, toluene, dioxane attemperatures ranging from 0° C. to 120° C., for 1 to 60 hours.

EXAMPLES ##STR195##

To 50 mg of diol 22 in 1 mL of dichloromethane was added Tosyl chloride(42.5 mg), 4,4-dimethylaminopyridine (6 mg), triethylamine (95 μl), andthe reaction was stirred for 12 hours at 23° C. The volatiles wereremoved in vacuo and the crude mixture (containing 165, thebis-tosylated compound and the corresponding epoxide) was separated byflash chromatography (ethyl acetate-hexane eluent) to give a mixture of165 and the corresponding bis-tosylated compound (27 mg total).##STR196##

The mixture consisting of 165 and the corresponding bis-tosylatedcompound was oxidized as described above for compound 24. The crudemixture was purified by flash chromatography (ethyl acetate-hexaneeluent) to give 3.9 mg of 166 and 16 mg of the correspondingbis-tosylate. ¹ H NMR of 166 (400 MHz, d₆ -acetone) δ1.5 (d, 18H), 2.4(s, 3H), 6.4 (d, 1H), 6.5 (d, 1H), 6.95 (s, 1H), 7.35 (d, 2H) 7.42 (d,2H), 7.5 (d, 1H), 7.6 (m, 3H), 7.7 (dd, 4H), 8 (d, 2H). ##STR197##

To 3.9 mg of 166 was added 3 mg of para-methoxythiobenzamide and 0.5 mlof toluene and the reaction was heated at 65° C. for 12 hours. Thesolvent was removed in vacuo and the crude mixture was purified by flashchromatography (ethyl acetate-hexane eluent) to give 1.8 mg of 167. ¹ HNMR of 167 (400 MHz, CDCl₃) δ1.5 (d, 18H), 3.8 (s, 3H), 6.3 (dd, 2H),6.9 (d, 2H), 7.35 (d, 2H), 7.4 (m, 4H), 7.55 (m, 4H), 7.9 (d, 2H).##STR198##

¹ H NMR of 168 (400 MHz, CD₃ OD) δ3.8 (s, 3H), 6.45 (dd, 2H), 7.0 (d,2H), 7.4 (d, 2H), 7.5-7.7 (m, 8H), 7.9 (d, 2H).

Biological Protocols

PTP-1B Gene Cloning and Protein Purification

The following procedure was conducted for recombinant production andpurification of protein tyrosine phosphatase PTP-1B, for use as asubstrate in PTPase inhibition assays.

A. Production of a PTP-1B cDNA

A human placental cDNA library was synthesized in a 50 ul reactioncontaining 1 ug human placental poly(A)⁺ mRNA (Clontech, Palo Alto,Calif.), 4 ul random hexamer primers, 8 ul of 10 mM dNTPs (Pharmacia,Piscataway N.J.), 1 ul (200 U/ul) Moloney murine leukemia virus reversetranscriptase (Gibco-BRL, Canada), 0.5 ul (26 U/ul) "RNAsin™" which is aribonuclease inhibitor (Promega, Madison Wis.), and 12 ul 5×buffer(Gibco-BRL). The synthesis reaction was incubated at 37° C. for one hourand then heat inactivated at 95° C. for five minutes.

A PTP-1B cDNA was amplified, using polymerase chain reaction (PCR), fromthe cDNAs synthesized as described above. More particularly, based onthe published sequence of PTB-1B, two PCR primers were synthesized toamplify a portion of the PTP-1B coding sequence known to encode a 321amino acid fragment containing the PTP-1B catalytic domain and havingPTPase activity. See Hoppe et al., Eur. J. Biochem., 223:1069-77 (1994);Barford, D., et al., J. Molec. Biol., 239:726-730 (1994); Chernoff etal., Proc. Natl. Acad. Sci. USA, 87:2735-2739 (1990); Charbonneau et al.Proc. Natl. Acad. Sci. USA, 86:5252-5256 (1989). The primers had thefollowing respective sequences:

PTP-1B-A(5') (SEQ ID NO: 1)

5'CGCACTGGATCCTCATGGAGATGGAAAAGG3'

PTP-1B-B(3') (SEQ ID NO: 2)

5'CTCCCTGAATTCCTAATTGTGTGGCTCCAGG3'

The first primer, which hybridizes to the non-coding strand, correspondsto the 5' portion of the PTP-1B coding sequence and encodes a BamH Irestriction site upstream of the initiation codon, to facilitatecloning. The second primer, which hybridizes to the coding strand,corresponds to the 3' portion of the PTB-1B fragment of interest, andencodes a stop codon and an EcoR I restriction site downstream from thestop codon.

A 100 μl PCR reaction mixture containing approx. 1 ug of the humanplacental cDNA library, 0.2 mM of each dNTP, 30 uM of each primer,1×Amplitaq DNA polymerase buffer (Perkin-Elmer, Norwalk Conn.), and 5units "Amplitaq™" which is a heat stable DNA polymerase (Perkin-Elmer)was denatured at 94° C. for 5 minutes and then subjected to 25 cycles ofamplification as follows: 1) 94° C. denaturation for 1 minute; 2) 55° C.annealing for 1 minute; and 3) 72° C. primer extension for 1 minute.

The PCR reaction product (992 bp) was digested with BamH I and EcoR I(New England Biolabs, Beverly Mass.) to yield a 975 bp product encodingthe 321 amino acid PTP-1B protein fragment, and having "sticky ends" tofacilitate cloning.

B. Production of a PTP-1B expression vector

The 975 bp PTP-1 B partial cDNA was purified by agarose gelelectrophoresis and ligated into a BamH I/EcoR I-digested pGEX-3X™ whichis an expression plasmid permitting amino-terminal fusion of proteinwith glutathione-S-transferase (GST) (Pharmacia, Piscataway, N.J.). ThepGEX vector is designed to produce a fusion of glutathione-S-transferase(GST) to a protein encoded by another DNA fragment inserted into thevector's cloning site. Complete sequencing of the insert of theresultant plasmid, designated pGEX™-3X™-PTP-1B, confirmed the identityof the PTP-1B cDNA, and insertion in the proper orientation and readingframe.

C. Expression and Purification of GST/PTP1B fusion protein

E. coli strain DH5α (Gibco-BRL) was transformed with plasmidpGEX-3X-PTP-1B following the supplier's transformation protocol andgrown at 37° C. with vigorous shaking in Luria-Bertani brothsupplemented with 100 ug/ml ampicillin. When the cultures reached anO.D.₆₀₀ of 0.7-1, production of the GST/PTP-1B fusion protein wasinduced with 0.1 mM IPTG (Isopropyl b-D-Thiogalactoside). After 3additional hours of culturing at 37° C., the bacteria were pelleted bycentrifugation.

The bacterial pellet was resuspended in 10×(w/v) lysis buffer consistingof 12.5 mM HEPES, 2 mM EDTA, pH 7.0, 15 mM b-mercaptoethanol (bME) and 1mM PMSF. The lysate was sonicated (on ice) until slight clearing wasobserved (approx. three min.) and then centrifuged at 10,000 revolutionsper minute (RPM) for 10 min. The supernatant was diluted 1:4 with bufferA (25 mM HEPES, pH 7.0, and 15 mM bME).

Primary purification was achieved using a 5 ml Hi-Trap pre-packed Qcolumn™ which is a pre-packed anion exchange column (Pharmacia). Afterloading the diluted supernatant onto the column, the column was washedwith 10 bed volumes of buffer A. The GST/PTP-1B fusion protein was theneluted using a linear gradient of Buffer A and Buffer B (buffer A+1MNaCl).

Eluted fractions containing protein were identified by SDS-PAGE andCoomassie Blue staining (Pharmacia PhastSystem), which is an integratedautomated system for running SDS-PAGE gels and fractions containingPTP-1B activity were identified using the PTP-1B activity assaydescribed below. Elution of the fusion protein occurred at about 30%Buffer B.

Fractions containing PTPase activity were pooled, diluted 1:4 with NETbuffer (20 mM Tris, pH 8.8, 100 mM NaCl, 1 mM EDTA and 15 mM bME), andloaded onto a 10 ml GST-Sepharose 4B column (Pharmacia). After loading,the column was washed first with 3 bed volumes of NET buffer+1% NP40(Sigma Chemical Co., St. Louis, Mo.), then with NET buffer until O.D. at280 nm was basal. The GST/PTP-1B fusion protein was eluted from thecolumn using 10 mM glutathione in 33 mM Tris, pH 8.0. Elution ofproteins was monitored at O.D.₂₈₀ and fractions were assayed foractivity and run on SDS-PAGE as described above. PTP-1B fusion proteineluted after approx. 4-5 minutes (flow rate 1 ml/min.).

The GST/PTP-1B-containing fractions from the GST-Sepharose 4B which isglutathione S-transferase (GST) coupled to sepharose beads purificationwere pooled, concentrated into a final storage buffer (0.2M NaCl, 25 mMHEPES, 1 mM EDTA, and 5 mM DTT, pH 7.0) using a 1 ml Hi-Trap Q™ which isa pre-packed anion exchange column (Pharmacia), and stored at -80° C.(final concentration of 0.52 mg/ml). The foregoing procedure yieldedapproximately 5 mg of PTP-1B fusion protein per 500 ml of culturedcells, purified to substantial homogeneity as assessed by SDS-PAGE.

Assay of PTP-1B Activity

PTP-1B enzymatic activity of samples was assayed in microtiter plates asfollows.

The protein concentration of the PTP-1B enzyme preparation wasdetermined using the Bio-Rad Protein Assay kit which is a kit containingreagents for determining protein concentrations (Bio-Rad, HerculesCalif.). An aliquot from each sample was taken and diluted to 2 mgprotein/ml using activity assay buffer (100 mM Sodium Acetate, pH 6.0, 1mM EDTA, 0.1% TX-100 (International Biotechnologies, Inc.) and 15 mMbME) to form a PTP-1B stock solution.

A 100 ul reaction mixture was prepared containing 10 ul of the PTP-1Bstock solution, 10 ul of 9 mM p-nitrophenylphosphate ((pNPP), SigmaChemical Co., St. Louis Mo.), and 80 ul of activity assay buffer (100 mMsodium acetate, pH 6.0, 1 mM EDTA, 0.1% Triton X-100™ Rohm & Hass Co.which is a detergent comprising isooctyl phenyl polyethoxy ethanol, 15mM bME). Reactions were mixed gently and incubated at 37° C. for 60minutes. Enzymatic cleavage of phosphate from pNPP (a tyrosine phosphateanalog) is marked by a calorimetric change in this substrate. See, e.g.,Imbert et al., Biochem J., 297:163-173 (1994); Ghosh and Miller,Biochem. Biophys. Res. Comm., 194:36-44 (1993); Zanke et al., Eur. J.Immunol., 22:235-39 (1992).

Reactions were stopped by addition of 10 ul of a 0.5M NaOH/50% EtOHsolution. To determine the enzymatic activity, absorbance readings ofthe reactions were measured at 405 nm using a Molecular DevicesThermomax Plate Reader™ which is a spectrophotometer (Menlo ParkCalif.).

CD45 Gene Cloning and Protein Purification

The following procedure was conducted for recombinant production andpurification of protein tyrosine phosphatase CD45, for use as asubstrate in PTPase inhibition assays.

A. Production of a CD45 cDNA, and production of a CD45 expression vector

A human cDNA library was synthesized from RNA isolated from the humanJurkat cell line, as described above for PTP-1B

CD45 cDNA was amplified, using polymerase chain reaction (PCR), from thecDNAs synthesized above. Two PCR primers were synthesized to amplify thecoding sequence of CD45. The primers had the following respectivesequences:

CD45 (5') (SEQ ID NO: 3)

5'CTACATCCCGGGATGTCCTGCAATTTAGATG 3'

CD45 (3') (SEQ ID NO: 4)

5'CATTTATGTCCCGGGCTATGAACCTTGAT 3'

The first primer corresponds to the 5' portion of the CD45 codingsequence and encodes a Sma I restriction site upstream of the initiationcodon, to facilitate cloning. The second primer corresponds to the 3'portion of the CD45 sequence, and encodes a stop codon and a Sma Irestriction site downstream from the stop codon.

The PCR reaction product (2127 bp) was digested with Sma I (New EnglandBiolabs, Beverly Mass.) to yield a 2110 bp product. The pET24C. plasmidvector (Novagen, Inc., Madison Wis.) was digested with the BamH Irestriction enzyme, and the "sticky" ends were filled using T4 DNApolymerase according to the manufacturer's instructions (New EnglandBiolabs, Beverly Mass.); the resulting plasmid DNA was ligated to SmaI-digested CD45 PCR product. The pET24C vector is designed to producehigh levels of the protein encoded by cDNA inserted into the vector'scloning site (CD45), in bacterial hosts. Complete sequencing of theinsert of the resultant plasmid, designated pET24C-CD45, confirmed theidentity of the CD45 cDNA, and insertion in the proper orientation andreading frame.

C. Expression and Purification of CD45 protein

E. coli strain DH5α (Gibco-BRL) was transformed with pET24C-CD45following the supplier's transformation protocol, plated ontoLuria-Bertani agar plates supplemented with 30 ug/ml kanamycin and grownovernight at 37° C. A single bacterial colony was transferred into 50mls Luria-Bertani broth supplemented with 30 ug/ml kanamycin and grownovernight with vigorous shaking. This overnight culture was split intotwo equal parts, and added to 2 L Luria-Bertani broth supplemented with50 ug/ml kanamycin. When the cultures reached an O.D.₆₀₀ of 1,production of the recombinant CD45 protein was induced with 0.1 mM IPTG(Isopropyl b-D-Thiogalactoside). After 5 additional hours of culturingat 37° C., the bacteria were pelleted by centrifugation.

The bacterial pellet (approximately 5 grams) was resuspended in 10×(w/v)lysis buffer consisting of 12.5 mM HEPES, 2 mM EDTA, pH 7.0, 15 mM bMEand 1 mM PMSF. The lysate was sonicated (on ice) until slight clearingwas observed (approx. three min.) and then centrifuged at 10,000revolutions per minute (RPM) for 10 min. The supernatant was filteredthrough 1 mm Wattman filter paper, and 9.7 grams (i.e., 194 grams/L) ofammonium sulfate were added to the solution on ice to precipitatesoluble proteins. After a 1 hour incubation on ice, the lysate was spunat 10,000 RPM for 30 min. at 4 C.; supernatant was removed, and anadditional 7.6 grams (i.e., 151 grams/L) of ammonium sulfate were added.The resulting pellet was resuspended in 3 mls of buffer B (33 mMimidazole-HCl pH 8.0, 2 mM EDTA, 10 mM bME, 0.002% PMSF) and stored onice. After another 1 hour incubation on ice, the spin supernatant withammonium sulfate was spun again at 10,000 RPM for 30 mins at 4 C. Theresulting pellet from the second centrifugation was resuspended in 2 mlsof buffer B. The two pellet solutions were pooled and dialyzed overnightagainst buffer B.

Secondary purification was achieved using a Mono-Q column. (Pharmacia).After loading the diluted supernatant onto the column, the column waswashed with 10 bed volumes of buffer B. The recombinant CD45 protein wasthen eluted using a linear gradient of Buffer B and Buffer C. (bufferB+1M NaCl). Eluted fractions containing protein were identified bySDS-PAGE and Coomassie Blue staining (Pharmacia PhastSystem), andfractions containing CD45 activity were identified using the CD45activity assay described below.

The CD45-containing fractions from the MonoQ column™ from Pharmaciawhich is an anion exchange column purification were pooled and stored at4 C.

Assay of CD45 Activity

CD45 enzymatic activity of samples was assayed in microtiter plates asfollows.

A 100 ul reaction mixture was prepared containing 10 ul of the CD45stock solution, 10 ul of 9.3 mM p-nitrophenylphosphate ((pNPP), SigmaChemical Co., St. Louis Mo.), and 80 ul of activity assay buffer (100 mMsodium acetate, pH 6.0, 1 mM EDTA, 0.1% Triton X-100™ from Rohm & Hasswhich is a detergent comprising isooctyl phenyl polyethoxy ethanol, 15mM bME). Reactions were mixed gently and incubated at 37° C. for 60minutes. Reactions were stopped by addition of 10 ul of a 0.5M NaOH/50%EtOH solution. To determine the enzymatic activity, absorbance readingsof the reactions were measured at 405 nm using a Molecular DevicesThermomax Plate Reader which is spectrophotometer (Menlo Park Calif.).

In vitro PTPase Inhibition Assay

The ability of the compounds of the present invention, such as thecinnamic acid derivative compounds of Example 2, to inhibit the PTPaseactivity of PTP-1B, CD45, PTP-1C, and PTPα was determined usingmodifications of the PTP-1B and CD45 activity assays described inExamples 3 and 4.

First, 0.001 mmol of the cinnamic acid derivative (or other PTPaseinhibitor compound) was dissolved in 100 ul of DMSO to create a 10 mMstock solution. The 10 mM stock solution was used to add varyingconcentrations (100 uM, 33 uM, 10 uM, 3 uM, 1 uM, 0.3 uM, 0.1 uM, 0.03uM, 0.01 uM or 0.003 uM) of the inhibitor compound to a series ofotherwise identical PTPase activity assay reactions (100 ul final volumein microtiter wells). Thus, each 100 ul reaction contained 10 ul PTPaseenzyme stock solution (final phosphatase concentration of approximately20 ng/well), 70 ul activity assay buffer, 10 ul pNPP stock solution(final pNPP concentration of 0.9 mM for PTP-1B assay, 0.93 mM for CD45assay, 0.5 mM for PTPα assay, and 8 mM for PTP-1C assay), and 10 ul ofthe diluted inhibitor compound in DMSO. Assay buffers contained: forCD45 and PTP-1B assays, 100 mM sodium acetate at pH 6.0, 1 mM EDTA, 0.1%Triton X-100™ which is a detergent from Rohm and Hass comprisingisooctyl phenyl polyethoxy ethanol and 15 mM bME; for PTP-1C. assays,100 mM sodium acetate at pH 5.5, 0.1% BSA, and 15 mM bME; for PTPαassays, 100 mM sodium acetate at pH 5.25, 0.1% BSA, and 15 mM bME.Purified phosphatase was added to the reaction mixtures to begin thereactions; the reactions were incubated at 37 C. for 60 min. (for PTP-1Band CD45 assays) or at 27 C. for 60 min. (for PTP-1C and PTPα assays),stopped, and colorimetrically analyzed as described above. As positiveand negative controls, reactions were performed containing 10 ul DMSOwith no inhibitor compound or containing the known PTPase inhibitorsvanadate (final concentration 0.5 mM; for PTP-1B and CD45 assays) orammonium molybdate (final concentration 1 mM; for PTP-1C and PTPαassays) substituted for the inhibitor compound of the invention.

The concentration of inhibitor compound required to inhibit 50% of thePTPase activity (IC50) was determined as follows. First, absorbancereadings from the negative control reactions were treated as a baselineand subtracted from the absorbance readings of the experimentalreactions. Then, for each reaction, a percent inhibition was calculatedusing the following formula:

    100 X 1-O.D..sub.405 reaction/O.D..sub.405 DMSO)!

For each inhibitor tested, an IC50 concentration was calculated from abest-fit computer analysis of the calculated percent inhibition for thevarious dilutions of the compound.

Inhibitor compounds having an IC50 less than 10 uM (and optimally lessthan 5 uM) for a particular PTPase were scored as highly effectiveinhibitors of that PTPase enzyme, and are preferred inhibitors of thepresent invention.

As it will be apparent to those persons skilled in the art, theforegoing biological data is not absolute and will vary according tomany factors such as assay conditions and the like.

                  TABLE 8                                                         ______________________________________                                                  % inhibition                                                                              % inhibition                                                                            % inhibition                                            of PTP1B    of PTPα                                                                           of PTP1C                                      Compound  at 1 μM  at 100 μM                                                                            at 100 μM                                  ______________________________________                                        36        52          0         42                                            37        85          63        59                                            38        93          71        63                                            39        82          47        53                                            40        88          82        62                                            41        39          20        17                                            42        84          92        88                                            43        76          82        79                                            44        79          87        86                                            45        85          85        84                                            46        75          73        61                                            47        68          48        63                                            48        69          3         33                                            49        37          0         35                                            50        50          37        25                                            ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        IC5O values (in μM) against PTP1B and CD45 for given compounds                  PTP1B   CD45       PTP1B CD45       PTP1B CD45                           ______________________________________                                        9    0.37    3.9    70  0.42  1.9   95   2     6                              13   31      --*    71  0.43  0.53  96   0.4   2.4                            23   0.27    --*    72  0.52  5.5   97   6     10                             25   0.89    --*    73  0.62  2.8   98   6     10                             27   0.5     --*    74  0.64  4.2   99   1.5   7.4                            29   0.8     --*    75  0.68  3.4   100  26    >100                           32   1.8     --*    76  0.68  0.93  133        --*                            54   0.072   0.73   77  0.78  7.5   134  3.4   20                             55   0.1     0.56   78  0.79  1.15  136  0.7   8                              56   0.135   0.94   79  4.8   8.2   140  1.2   20                             57   0.25    1.0    80  10    20    144  3     --*                            58   0.25    0.97   81  26    19    146  5.9   --*                            59   0.25    0.35   82  11.9  12.8  148  9     --*                            60   0.29    1.0    83  1.3   1.5   152  0.85  1.2                            61   0.97    0.955  84  1.2   2.7   153  2.65  1.91                           62   1.5     0.985  85  1.5   1.8   154  3.83  2.45                           63   1.7     2.4    86  1.8   7.1   155  1     1.3                            64   3.0     6.4    87  1.0   1.1   156  1.7   1.3                            65   1.3     1.4    88  2.65  7.8   157  5.5   1.5                            66   1.7     2.5    89  13.7  >100  160  0.98  1.52                           67   1.0     1.25   90  0.86  1.12  162  1.8   --*                            68   0.3     0.865  91  25.9  >100  168  3     --*                            69   0.41    1.9    94  0.7   7                                               ______________________________________                                         *--data not available                                                    

                  TABLE 10                                                        ______________________________________                                                       % inhibition                                                                            % inhibition                                                        of PTP1B  of CD45                                              Compound       at 1 μM                                                                              at 1 μM                                           ______________________________________                                        103            44%       14%                                                  104            24%       14%                                                  105            61%       18%                                                  106            45%       21%                                                  107            25%       51%                                                  108            30%       62%                                                  109            --*       14%                                                  110            --*       22%                                                  111            --*       18%                                                  112            --*       16%                                                  113            --*       61%                                                  ______________________________________                                         *--data not available                                                    

                  TABLE 11                                                        ______________________________________                                                  % inhibition          % inhibition                                            of PTP1B              of PTP1B                                      Compound  at 1 μM  Compound  at 1 μM                                    ______________________________________                                        116       41%         123       85%                                           117       67%         124       83%                                           118       56%         125       93%                                           119       71%         126       59%                                           120       67%         127       79%                                           121       73%         128       80%                                           122       87%                                                                 ______________________________________                                    

The compounds of the present invention have asymmetric centers and mayoccur as racemates, racemic mixtures, and as individual enantiomers ordiastereoisomers, with all isomeric forms being included in the presentinvention as well as mixtures thereof.

Pharmaceutically acceptable salts of the compounds of Formula (A1) thru(A11) where a basic or acidic group is present in the structure, arealso included within the scope of this invention. When an acidicsubstituent is present, such as --COOH, there can be formed theammonium, sodium, potassium, calcium salt, and the like, for use as thedosage form. When a basic group is present, such as amino or a basicheteroaryl radical, such as pyridyl, an acidic salt, such ashydrochloride, hydrobromide, acetate, maleate, pamoate,methanesulfonate, p-toluenesulfonate, and the like, can be used as thedosage form.

Also, in the case of the --COOH being present, pharmaceuticallyacceptable esters can be employed, e.g., methyl, tert-butyl,pivaloyloxymethyl, and the like, and those esters known in the art formodifying solubility or hydrolysis characteristics for use as sustainedrelease or prodrug formulations.

In addition, some of the compounds of the instant invention may formsolvates with water or common organic solvents. Such solvates areencompassed within the scope of the invention.

The term "therapeutically effective amount" shall mean that amount ofdrug or pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal, or human that is being sought by aresearcher, veterinarian, medical doctor or other clinician. Generally,a daily dose of about 0.5 mg/Kg to 100 mg/Kg body weight in divideddoses is suggested to treat PTPase related diseases. Such dosage has tobe individualized by the clinician.

The present invention also has the objective of providing suitabletopical, oral, and parenteral pharmaceutical formulations for use in thenovel methods of treatment of the present invention. The compounds ofthe present invention may be administered orally as tablets, aqueous oroily suspensions, lozenges, troches, powders, granules, emulsions,capsules, syrups or elixirs. The composition for oral use may containone or more agents selected from the group of sweetening agents,flavouring agents, colouring agents and preserving agents in order toproduce pharmaceutically elegant and palatable preparations. The tabletscontain the acting ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, (1) inertdiluents, such as calcium carbonate, lactose, calcium phosphate orsodium phosphate; (2) granulating and disintegrating agents, such ascorn starch or alginic acid; (3) binding agents, such as starch, gelatinor acacia; and (4) lubricating agents, such as magnesium stearate,stearic acid or talc. These tablets may be uncoated or coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. Coating may also beperformed using techniques described in the U.S. Pat. Nos. 4,256,108;4,160,452; and 4,265,874 to form osmotic therapeutic tablets for controlrelease.

Formulations for oral use may be in the form of hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin. They may alsobe in the form of soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, such as peanut oil, liquid paraffinor olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspension. Suchexpicients may be: (1) suspending agent such as sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;

(2) dispersing or wetting agents which may be (a) naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

Compounds of Formula (A1) thru (A11) may also be administered in theform of suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperature butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

The compounds of the present invention may also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compounds of Formula (A1) thru (A11) are employed.

What is claimed is:
 1. A compound with the structure depicted in Formula(A6): ##STR199## wherein at least one of R₁, R₂, R₃ and R₄ substituentshas the general structure depicted in Formula (B)

    X--C(R')═C(R")COOR'"                                   (B)

wherein (i) R' and R" are independently selected from the groupconsisting ofhydrogen, halo, cyano, nitro, trihalomethyl, C₁₋₁₁ alkyl,optionally substituted arylC₁₋₁₁ alkyl wherein the aryl substituents areindependently selected from the group consisting of hydrogen, alkoxy,halo, nitro, cyano, trihalomethyl, hydroxypyronyl, C₁₋₁₁ alkyl,arylC₁₋₁₁ alkyl, C₀₋₁₁ alkyloxyC₀₋₁₁ alkyl, arylC₀₋₁₁ alkyloxyC₀₋₁₁alkyl, C₀₋₁₁ alkylthioC₀₋₁₁ alkyl, arylC₀₋₁₁ alkylthioC₀₋₁₁ alkyl, C₀₋₁₁alkylaminoC₀₋₁₁ alkyl, arylC₀₋₁₁ alkylaminoC₀₋₁₁ alkyl, di(arylC₁₋₁₁alkyl)aminoC₀₋₁₁ alkyl, C₁₋₁₁ alkylcarbonylC₀₋₁₁ alkyl, arylC₁₋₁₁alkylcarbonylC₀₋₁₁ alkyl, C₁₋₁₁ alkylcarboxyC₀₋₁₁ alkyl, arylC₁₋₁₁alkylcarboxyC₀₋₁₁ alkyl, C₁₋₁₁ alkylcarbonylaminoC₀₋₁₁ alkyl, arylC₁₋₁₁alkylcarbonylaminoC₀₋₁₁ alkyl, --C₀₋₁₁ alkylCOOR₅, --C₀₋₁₁ alkylCONR₆ R₇wherein R₅, R₆ and R₇ are independently selected from hydrogen, C₁₋₁₁alkyl, arylC₀₋₁₁ alkyl, or R₆ and R₇ are taken together with thenitrogen to which they are attached forming a cyclic system containing 3to 8 carbon atoms with at least one C₁₋₁₁ alkyl, arylC₀₋₁₁ alkylsubstituent. (ii) R'" is selected from the group consisting of(a)hydrogen, (b) C₁₋₁₁ alkyl, substituted C₁₋₁₁ alkyl wherein thesubstituents are independently selected from halo, cyano, nitro,trihalomethyl, carbamoyl, tetrahydrofuryl, pyrrolidinyl, piperidinyl,morpholinyl, piperazinyl, hydroxypyronyl, C₀₋₁₁ alkyloxy, arylC₀₋₁₁alkyloxy, C₀₋₁₁ alkylthio, arylC₀₋₁₁ alkylthio, C₀₋₁₁ alkylamino,arylC₀₋₁₁ alkylamino, di(arylC₀₋₁₁ alkyl)amino, C₁₋₁₁ alkylcarbonyl,arylC₁₋₁₁ alkylcarbonyl, C₁₋₁₁ alkylcarboxy, arylC₁₋₁₁ alkylcarboxy,C₁₋₁₁ alkylcarbonylamino, aryl C₁₋₁₁ alkylcarbonylamino, --C₀₋₁₁alkylCOOR₈, --C₀₋₁₁ alkylCONR₉ R₁₀ wherein R₈, R₉ and R₁₀ areindependently selected from hydrogen, C₁₋₁₁ alkyl, arylC₀₋₁₁ alkyl, orR₉ and R₁₀ are taken together with the nitrogen to which they areattached forming a cyclic system containing 3 to 8 carbon atoms with atleast one C₁₋₁₁ alkyl, arylC₀₋₁₁ alkyl substituent, (c) mono-, di- andtri-substituted arylC₀₋₁₁ alkyl wherein the aryl substituents aredefined as above for R'and R", (iii) X is a mono-, di- or trisubstitutedaryl wherein the aryl substituents are defined as above for R' and R",and aryl is selected from phenyl, biphenyl, naphthyl, dihydronaphthyl,tetrahydronaphthyl, indenyl, indanyl, azulenyl, anthryl, phenanthryl,fluorenyl, pyrenyl, thienyl, benzothienyl, isobenzothienyl,2,3-dihydrobenzothienyl, furyl, pyranyl, benzofuranyl, isobenzofuranyl,2,3-dihydrobenzofuranyl, pyrrolyl, indolyl, isoindolyl, indolizinyl,indazolyl, imidazolyl, benzimidazolyl, pyridyl, pyrazinyl, pyradazinyl,pyrimidinyl, triazinyl, quinolyl, isoquinolyl, 4H-quinolizinyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, chromanyl, benzodioxolyl, piperonyl,purinyl, hydroxypyronyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl,isothiazolyl, benzthiazolyl, oxazolyl, isoxazolyl, benzoxazolyl,oxadiazolyl, or thiadiazolyl,and wherein the remaining of R₁, R₂, R₃ andR₄ are independently selected from the group consisting of: (i)hydrogen; (ii) C₁₋₁₁ alkyl, substituted C₁₋₁₁ alkyl wherein the alkylsubstituents are defined as above, (iii) arylC₀₋₁₁ alkyl, (iv) mono-,di- and tri-substituted arylC₀₋₁₁ alkyl wherein the aryl substituentsare defined as above, with the proviso that when R₃ and R₄ are selectedfrom substituted phenyl or substituted furyl then the phenyl and furylsubstituents exclude hydroxy, halo, trifluoromethyl, C₁₋₆ alkyl, C₁₋₆alkyloxy, C₁₋₆ alkylthio, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino,phenylC₁₋₆ alkylamino and di(phenylC₁₋₆ alkyl)amino, or itspharmaceutically acceptable salts or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.2. A compound as defined in claim 1 with the structure as depicted below##STR200## or its pharmaceutically acceptable salts, or from Rohm & Hasswhich is a detergent comprising isooctyl phenyl polyethoxy ethanolesters, thereof.
 3. A compound as defined in claim 1 with the structureas depicted below ##STR201## or its pharmaceutically acceptable salts,or from Rohm & Hass which is a detergent comprising isooctyl phenylpolyethoxy ethanol esters, thereof.
 4. A compound as defined in claim 1with the structure as depicted below ##STR202## or its pharmaceuticallyacceptable salts, or from Rohm & Hass which is a detergent comprisingisooctyl phenyl polyethoxy ethanol esters, thereof.
 5. A compound asdefined in claim 1 with the structure as depicted below ##STR203## orits pharmaceutically acceptable salts, or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.6. A compound as defined in claim 1 with the structure as depicted below##STR204## or its pharmaceutically acceptable salts, or from Rohm & Hasswhich is a detergent comprising isooctyl phenyl polyethoxy ethanolesters, thereof.
 7. A compound as defined in claim 1 with the structureas depicted below ##STR205## or its pharmaceutically acceptable salts,or from Rohm & Hass which is a detergent comprising isooctyl phenylpolyethoxy ethanol esters, thereof.
 8. A compound as defined in claim 1with the structure as depicted below ##STR206## or its pharmaceuticallyacceptable salts, prodrugs, esters, or solvates thereof.
 9. A compoundas defined in claim 1 with the structure as depicted below ##STR207## orits pharmaceutically acceptable salts, or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.10. A compound as defined in claim 1 with the structure as depictedbelow ##STR208## or its pharmaceutically acceptable salts, or from Rohm& Hass which is a detergent comprising isooctyl phenyl polyethoxyethanol esters, thereof.
 11. A compound as defined in claim 1 with thestructure as depicted below ##STR209## or its pharmaceuticallyacceptable nsalts, prodrugs, esters, or solvates thereof.
 12. A compoundas defined in claim 1 with the structure as depicted below ##STR210## orits pharmaceutically acceptable salts, or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.13. A compound as defined in claim 1 with the structure as depictedbelow ##STR211## or its pharmaceutically acceptable salts, or from Rohm& Hass which is a detergent comprising isooctyl phenyl polyethoxyethanol esters, thereof.
 14. A compound as defined in claim 1 with thestructure as depicted below ##STR212## or its pharmaceuticallyacceptable salts, or from Rohm & Hass which is a detergent comprisingisooctyl phenyl polyethoxy ethanol esters, thereof.
 15. A compound asdefined in claim 1 with the structure as depicted below ##STR213## orits pharmaceutically acceptable salts, or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.16. A compound as defined in claim 1 with the structure as depictedbelow ##STR214## or its pharmaceutically acceptable salts, or from Rohm& Hass which is a detergent comprising isooctyl phenyl polyethoxyethanol esters, thereof.
 17. A compound as defined in claim 1 with thestructure as depicted below ##STR215## or its pharmaceuticallyacceptable salts, or from Rohm & Hass which is a detergent comprisingisooctyl phenyl polyethoxy ethanol esters, thereof.
 18. A compound asdefined in claim 1 with the structure as depicted below ##STR216## orits pharmaceutically acceptable salts, or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.19. A compound as defined in claim 1 with the structure as depictedbelow ##STR217## or its pharmaceutically acceptable salts, or from Rohm& Hass which is a detergent comprising isooctyl phenyl polyethoxyethanol esters, thereof.
 20. A compound as defined in claim 1 with thestructure as depicted below ##STR218## or its pharmaceuticallyacceptable salts, or from Rohm & Hass which is a detergent comprisingisooctyl phenyl polyethoxy ethanol esters, thereof.
 21. A compound asdefined in claim 1 with the structure as depicted below ##STR219## orits pharmaceutically acceptable salts, or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.22. A compound as defined in claim 1 with the structure as depictedbelow ##STR220## or its pharmaceutically acceptable salts, or from Rohm& Hass which is a detergent comprising isooctyl phenyl polyethoxyethanol esters, thereof.
 23. A compound as defined in claim 1 with thestructure as depicted below ##STR221## or its pharmaceuticallyacceptable salts, or from Rohm & Hass which is a detergent comprisingisooctyl phenyl polyethoxy ethanol esters, thereof.
 24. A compound asdefined in claim 1 with the structure as depicted below ##STR222## orits pharmaceutically acceptable salts, or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.25. A compound as defined in claim 1 with the structure as depictedbelow ##STR223## or its pharmaceutically acceptable salts, or from Rohm& Hass which is a detergent comprising isooctyl phenyl polyethoxyethanol esters, thereof.
 26. A compound as defined in claim 1 with thestructure as depicted below ##STR224## or its pharmaceuticallyacceptable salts, or from Rohm & Hass which is a detergent comprisingisooctyl phenyl polyethoxy ethanol esters, thereof.
 27. A compound asdefined in claim 1 with the structure as depicted below ##STR225## orits pharmaceutically acceptable salts, or from Rohm & Hass which is adetergent comprising isooctyl phenyl polyethoxy ethanol esters, thereof.28. A compound as defined in claim 1 with the structure as depictedbelow ##STR226## or its pharmaceutically acceptable salts, or from Rohm& Hass which is a detergent comprising isooctyl phenyl polyethoxyethanol esters, thereof.
 29. A compound as defined in claim 1 with thestructure as depicted below ##STR227## or its pharmaceuticallyacceptable salts, or from Rohm & Hass which is a detergent comprisingisooctyl phenyl polyethoxy ethanol esters, thereof.
 30. A compound aswith the structure as depicted below ##STR228## or its pharmaceuticallyacceptable salts, or from Rohm & Hass which is a detergent comprisingisooctyl phenyl polyethoxy ethanol esters, thereof.
 31. A compound asdefined in claim 1 with the structure as depicted below ##STR229## orits pharmaceutically acceptable salts, prodrugs, esters, or solvatesthereof.
 32. A pharmaceutical composition comprising as active componenta compound according to a compound selected from the group consisting ofthose defined in claim 1 together with a pharmaceutically acceptablecarrier or diluent.
 33. A pharmaceutical composition suitable formodulating the activity of PTPases or other molecules with tyrosinephosphate recognition unit(s) comprising an effective amount of acompound according to a compound selected from the group consisting ofthose defined in claim 1 together with a pharmaceutically acceptablecarrier or diluent.
 34. The pharmaceutical composition according to anyone of the claims 32 or 33 suitable for treating type I diabetes, typeII diabetes, impaired glucose tolerance, insulin resistance, obesity,immune dysfunctions, autoimmunity, AIDS, diseases with dysfunctions ofthe coagulation system, allergic diseases, osteoporosis, proliferativedisorders of cancer and psoriasis, diseases with decreased or increasedsynthesis or effects of growth hormone, diseases with decreased orincreased synthesis of hormones or cytokines that regulate the releaseof/or response to growth hormone, Alzheimer's disease, schizophrenia orinfectious diseases.
 35. The pharmaceutical composition according to anyone of the claims 32, 33 or 34 comprising between 0.5 mg and 1000 mg ofa compound according to a compound selected from the group consisting ofthose defined in claim 1 per unit dose.
 36. A method of modulating theactivity of PTPases or other molecules with phosphotyrosine recognitionunit(s) in a subject in need of such treatment comprising administeringto said subject an effective amount of a compound or compositionaccording to a compound selected from the group consisting of thosedefined in claim
 1. 37. An immobilized compound comprising a suitablesolid-phase coupled with a compound according to a compound selectedfrom the group consisting of those defined in claim
 1. 38. A method forcoupling a compound according to a compound selected from the groupconsisting of those defined in claim 1 to a suitable solid-phase matrix.39. A method for isolating a protein or a glycoprotein with affinity fora compound according to a compound selected from the group consisting ofthose defined in claim 1 from a biological sample, comprisingcontactingan immobilized compound according to claim 37 with said biologicalsample in order for said immobilized compound to form a complex bybinding said protein or glycoprotein removing unbound material from saidbiological sample and isolating said complex extracting said protein orglycoprotein from said complex.
 40. A method for isolating aprotein-tyrosine phosphatase with affinity for a compound according to acompound selected from the group consisting of those defined in claim 1from a biological sample, comprisingcontacting an immobilized compoundaccording to claim 37 with said biological sample in order for saidimmobilized compound to form a complex by binding said protein-tyrosinephosphatase removing unbound material from said biological sample andisolating said complex extracting said protein-tyrosine phosphatase fromsaid complex.
 41. A method for isolating a Src-homology 2 domaincontaining protein or a phosphotyrosine binding domain containingprotein with affinity for a compound according to a compound selectedfrom the group consisting of those defined in claim 1 from a biologicalsample, comprisingcontacting an immobilized compound according to claim37 with said biological sample in order for said immobilized compound toform a complex by binding said Src-homology 2 domain containing proteinor a phosphotyrosine binding domain containing protein removing unboundmaterial from said biological sample and isolating said complexextracting said Src-homology 2 domain containing protein or aphosphotyrosine binding domain containing protein from said complex.